TW201918494A - Bifunctional compounds - Google Patents

Abstract

The application discloses compounds useful in treatment of diabetes, weight loss and/or reduction of cardiovascular risks. The compounds are bi-functional and therefore suitable as a simple treatment for patients that may benefit from treatment with both a GLP-1 receptor agonist and a PCSK9 inhibitor.

Description

   Bifunctional compound   

The present invention relates to a bifunctional compound that inhibits PCSK9 and stimulates the GLP-1 receptor, and the medical use thereof.

High LDL-C (low density lipoprotein cholesterol) levels and dyslipidemia are recognized causes of cardiovascular disease.

Statin has been approved for the treatment of dyslipidemia for 25 years. This type has demonstrated a substantial and consistent reduction in cardiovascular events and an acceptable safety profile. Best-selling statin Atorvastatin (Lipitor ^™ ) is by far the best-selling drug in the world, with sales of more than $ 125 billion between 1996 and 2012.

Although statins and other lipid-lowering agents are available and widely used, many patients fail to meet their target LDL-C levels and are still at high risk of developing cardiovascular disease. PCSK9 (preprotein-converting enzyme subtilisin / Kexin type 9) promotes the degradation of liver LDL-R (LDL receptor), thereby reducing liver LDL-R surface appearance and therefore the clearance of LDL particles. In contrast, blocking PCSK9 increased clearance of LDL-C and other atherogenic lipoproteins. Indeed, LDL receptors contribute to the clearance of atherogenic lipoproteins other than LDL, such as medium density lipoproteins and residual microparticles. Increased medium density lipoprotein and residual particulate clearance may have therapeutic benefits other than by the LDL reduction provider.

Statin increases the performance of both LDL-R and PCSK9 by the SREBP2 transcription factor. The increased performance of PCSK9 can reduce the efficacy of statin on LDL-C self-circulation clearance. By inhibiting the binding of PCSK9 to LDL-R and thereby preventing LDL-R degradation, the effectiveness of statin is improved. In conclusion, PCSK9 inhibition provides a novel approach to lipid control.

The EGF (A) (epidermal growth factor-like domain A) sequence (40 amino acids) of LDL-R (LDL-R- (293-332)) is widely accepted as a site for PCSK9 binding. It has been shown isolated wild type EGF (A) peptide will be low μM range of IC ₅₀ inhibition of PCSK9 binding to the LDL-R (Biochemical and Biophysical Research Communications 375 ( 2008) 69-73). This poor potency would prevent the viable medical use of the EGF (A) peptide. In addition, the half-life of such peptides would be expected to be too short for therapeutic use.

WO2012177741 and J. Mol. Biol. (2012) 422,685-696 disclose analogs of EGF (A) and Fc fusions thereof.

Two anti-PCSK9 antibodies, alirocumab / Praluent® and evolocumab / Repatha®, have recently been approved for the treatment of high LDL-C levels. These are administered every two weeks by 1 ml subcutaneous injection.

In WO 2015/127273, a fusion of an anti-PCSK9 antibody and a GLP-1 agonist was studied to find the functionality of a combination of GLP-1 and anti-PCSK9 antibodies.

There are multiple treatments for the treatment of diabetes and cardiovascular disease, but the combination of multiple independent drugs is not always attractive and a single molecule that addresses both disease states is useful for improving treatments such as efficacy, compliance, and convenience ) Will be what you want.

The present invention relates to EGF (A) analogs having the ability to inhibit the binding of PCSK9 to LDL-R and thereby reduce LDL cholesterol. Such molecules can be combined with GLP-1 receptor agonists to form bifunctional molecules that provide further therapeutic options (solving both diabetes and cardiovascular disease with a single drug). The invention relates in one aspect to compounds comprising a GLP-1 agonist and a PCSK9 inhibitor.

One aspect of the invention pertains to compounds comprising GLP-1 analogs and EGF (A) analogs, wherein i. The GLP-1 analog is GLP-1 (7-37) identified by SEQ ID NO: 137 And ii. The EGF (A) analog is an analog of the EGF (A) domain (293-332) of LDL-R identified by SEQ ID No: 1.

In one embodiment, such a compound may comprise a fusion polypeptide comprising the two analogs, if necessary, linked through a spacer peptide inserted between the two analogs.

The compounds may further include a half-life extending portion, which may be considered to be a substituent attached to the amino acid residue of the GLP-1 analog, the EGF (A) analog, or one of the spacers. In one embodiment, the compound comprises one or two substituents attached to different amino acid residues of the fusion polypeptide.

In a further aspect, the invention relates to a pharmaceutical composition comprising a compound of the invention and a medical use of a compound of the invention.

The present invention relates to bifunctional compounds that stimulate GLP-1 receptors and inhibit PCSK9. In order to prepare pharmaceutically relevant compounds, modifications to wild-type peptides are needed to improve both functionality and enable convenient administration.

One aspect of the invention relates to compounds comprising a GLP-1 receptor agonist and a PCSK9 inhibitor. Several GLP-1 receptor agonists are known in the art and can be combined with various PCSK9 inhibitors. As described herein, the GLP-1 receptor agonist can be an analog of human GLP-1 (7-37) (SEQ ID No: 137) or such as is also known to act as a GLP-1 receptor Agent acting extendin 4 and its analogs.

PCSK9 inhibitors are known in the form of antibodies, and this application is mainly related to PCSK9 inhibition in the form of analogs of the EGF (A) domain (293-332) (SEQ ID NO: 1) of LDL-R Agent.

One aspect of the present invention pertains to compounds comprising a GLP-1 analog and an EGF (A) analog, wherein the GLP-1 analog is an analog of GLP-1 (7-37) (SEQ ID No: 137) and The EGF (A) analog is an analog of the EGF (A) domain (293-332) (SEQ ID NO: 1) of LDL-R.

The term "compound" is used herein to refer to molecular entities, and "compound" may therefore have different structural elements other than the smallest element defined for each compound or group of compounds. Thus, a compound can be a polypeptide or a derivative thereof, so long as the compound contains a defined structural and / or functional element.

The term "compound" is also intended to encompass its pharmaceutically relevant form, that is, the invention relates to a compound or a pharmaceutically acceptable salt, amidine, or ester thereof as defined herein.

The term "peptide" or "polypeptide" (e.g., as used herein before and after) refers to a compound that contains a series of amino acids that are linked to each other through a amide (or peptide) bond. In a particular embodiment, the peptide is composed of amino acids connected to each other through peptide bonds.

The terms "fused" and "fused" are used for polypeptides comprising two individually defined peptide sequences linked by peptide bonds or linked by peptide spacers (also linked by peptide bonds). Fusion polypeptides are therefore continuous fragments of amino acid residues linked by peptide bonds.

The term "analog" generally refers to a peptide whose sequence has one or more amino acid changes when compared to a reference amino acid sequence. Certain analogues that "contain" certain changes may include further changes when compared to their equivalent reference sequences. In particular embodiments, the analogues “have” or “comprise” the changes specifically specified. In other particular embodiments, the analogues are "consisting of" such changes. When the term "consist" or "consisting" is used with respect to an analog (e.g., the analog is composed of a specified amino acid substitution or similar formed by a specified amino acid substitution )), It should be understood that the specified amino acid substitutions are the only amino acid substitutions in the analog. Conversely, a "comprising" group of specified amino acid substituted analogs may have additional substitutions. "Analogs" may also include elongation of the amino acid at the N-terminal and / or C-terminal positions and / or truncation at the N-terminal and / or C-terminal positions.

In general, amino acid residues can be identified by their full name, their one-letter code, and / or their three-letter code. These three methods are completely equivalent.

An amino acid is a molecule containing an amine group and a carboxylic acid group, and (if necessary) one or more additional groups (often referred to as side chains).

The term "amino acid" includes proteinogenic (or natural) amino acids (including 20 standard amino acids), as well as non-proteinogenic (or non-natural) amino acids. Proteinogenic amino acids are those which are naturally incorporated into proteins. Standard amino acids are those encoded by the genetic code. Non-proteinogenic amino acids are those that are not found in the protein, or are not manufactured by standard cellular machinery (eg, they may have undergone post-translational modifications). Non-limiting examples of non-proteinogenic amino acids are Aib (α-aminoisobutyric acid, or 2-aminoisobutyric acid), n-leucine, n-valine, and protein-forming amino acids. D-isomer.

In the following, the amino acid of each of the peptides of the present invention for which the optical isomers are not specified shall be understood to mean the L-isomers (unless otherwise specified).

GLP-1 analogs

The present invention relates to compounds comprising a glucagon-like peptide 1 (GLP-1) analog. The term "GLP-1 analog" is used herein to refer to an analog (or variant) of the human glucagon-like peptide-1 (GLP-1 (7-37)), the sequence of which is given in SEQ ID NO : 137 is included in the Sequence Listing. The peptide having the sequence of SEQ ID NO: 137 may also be referred to as "native" or wild-type GLP-1.

The numbering of amino acid residues in the GLP-1 analogues of the present invention (such as "position 8") is in accordance with the practice in the art for natural GLP-1, that is, the first (N-terminal) amino group The acid residues are numbered or assigned position no. 7, and the subsequent amino acid residues downstream toward the C-terminus are numbered 8, 9, 10, and so on, until the final (C-terminal) amino acid residue base. In natural GLP-1, the C-terminal amino acid residue is Gly, number 37.

Numbering is done in different ways in the sequence listing, where the first amino acid residue (His) of SEQ ID NO: 137 is designated as no.1, and the last amino acid residue (Gly) is designated as no.31. However, in this article, we follow the numbering practices established in the technical field to which we belong, as explained above.

GLP-1 analogs are known in the art and several GLP-1 analogs are supplied to the market for the treatment of type 2 diabetes and obesity. As described above, the GLP-1 analog is a variant of the wt human GLP-1 sequence and therefore contains one or more amino acid substitutions, deletions, and / or additions compared to SEQ ID NO.137.

Each of these GLP-1 analogs can be described by referring to: i) the number of the amino acid residues in the natural GLP-1 (7-37) corresponding to the amino acid residues being changed ( (I.e. the corresponding position in natural GLP-1), and ii) the actual change.

In other words, the GLP-1 analogues of the present invention can be described by referring to natural GLP-1 (7-37) peptides, that is, as some of the amino acid residues are equivalent to natural GLP-1 (7-37) (SEQ ID NO: 137) A variant that has been changed by comparison.

These changes may (independently) manifest one or more amino acid substitutions, additions, and / or deletions.

The following are non-limiting examples of suitable analog nomenclature. The GLP-1 analogue incorporated as GLP-1 analogue # 2 (SEQ ID NO: 139) and included in compound # 1 can be referred to as (8Aib, 34R) GLP-1 (7-37).

When this analog is aligned with natural GLP-1, the amino acid Aib at the position corresponding to position 8 in natural GLP-1 (based on the alignment) is located in the analog The amino acid R corresponding to the position 34 in the natural GLP-1, and all other amino acids in this analog are identical to the corresponding amino acid in the natural GLP-1.

When compared to wt GLP-1 (SEQ ID NO: 137), analogs "comprising" certain specified changes may contain further changes. In contrast, the term "composition" is used to refer to a particular embodiment in which the analog has only the specifically stated changes, i.e. the GLP-1 is similar when compared to wt GLP-1 (SEQ ID NO: 137) There are no further changes. By referring back to the above example, GLP-1 analog # 2 (SEQ ID NO: 139) can be said to be a GLP-1 analog in which its substitution consists of 8Aib and 34R, or 8Aib and 34R for short The composed GLP-1 analogue.

The expression "position equivalent to" or "corresponding position" is used herein to define a variant by referring to a reference sequence such as natural GLP-1 (7-37) (SEQ ID NO: 137). Characteristics of the altered position in the GLP-1 (7-37) sequence. Equivalent or corresponding positions (and the number of changes) are easily inferred, for example, by simple handwriting and visual inspection; and / or standard protein or peptide alignment programs such as "alignment based on Needleman-Wunsch algorithm" ". This algorithm is described in Needleman, SB and Wunsch, CD, (1970), Journal of Molecular Biology, 48: 443-453, and Myers and W. Miller in "Optimal Alignments in Linear Space" CABIOS (computer applications in the biosciences) (1988) 4: 11-17. For comparison, the preset scoring matrix BLOSUM62 and the preset consistency matrix can be used, and the penalty of the first residue in the gap can be set to -12, or preferably -10, and the gap in the gap can be additionally The penalty for residues is set at -2, or preferably at -0.5.

An example of such an alignment of the natural GLP-1 of SEQ ID NO: 137 and its analog identified by SEQ ID NO: 139 is inserted as follows:

When 6 is added to the position number shown in this alignment (for example, in SEQ ID NO 137 to "1" and "31"), the position number used in this article will be obtained. For example, in wt GLP-1 (which is exactly the same as SEQ ID NO: 137), the N-terminal amino acid (H) has position number 7 and the C-terminal amino acid (G) has number 37. Regarding GLP-1 analog # 2 (SEQ ID NO 139), the N-terminal amino acid (H) has the number 7 and the C-terminal amino acid (G) has the number 37 (like wt GLP-1) and residues 2 and 28 is replaced and numbered 8 and 34 respectively.

In the case of sequences that include special amino acid residues or the like (such as 2-amino-2-methylpropanoic acid (Aib) without single-letter codons) in the sequence, these may (for comparison purposes) be ( For example) X replacement. If desired, X can be manually corrected later.

The following are non-limiting examples of inferences that can be inferred from the above alignments: As an example, it can be inferred that sequence 2 has two amino acid changes compared to sequence 1 (that is, a period (". ”), A colon (": "), or a horizontal hyphen ("-")).

In the following, all amino acids of the GLP-1 analogues of the present invention for which optical isomers are not specified shall be understood to mean L-isomers (unless otherwise specified).

In one embodiment, the GLP-1 analog of the present invention is an analog of GLP-1 (7-37) consisting of 26 to 36 amino acid residues.

In one embodiment, GLP-1 analogs have up to 10 amino acid substitutions compared to human GLP-1 (7-37). In a further embodiment, GLP-1 analogs have up to 8 (such as up to 7, 6, 5, 4, 3, or 2) amino acid substitutions compared to human GLP-1 (7-37).

A large number of GLP-1 analogs and their functions as GLP-1 receptor agonists have been previously described.

The wt GLP-1 peptide of SEQ ID NO: 137 contains two Lys residues at positions 26 and 34. As can be seen below herein, these Lys residues are particularly relevant when it is intended to prepare a compound comprising a substituent attached via a Lys residue.

In one embodiment, a GLP-1 analog according to the invention comprises zero, one or two Lys residues. In one embodiment, the GLP-1 analog comprises one or two Lys residues, which are selected from wt Lys residues and Lys residues introduced into the GLP-1 analog by amino acid substitution. The Lys residue introduced by amino acid substitution can be considered as another Lys residue. In one embodiment, the GLP-1 analog comprises additional Lys residues. Additional Lys can be introduced into various positions in the GLP-1 analog, such as one or more positions selected from positions 12, 21, 23, 24, 25, 27, 30, 31, 32, 33, and 36K. In one embodiment, the GLP-1 analog comprises additional Lys selected from the group of 12K, 21K, 23K, 24K, 25K, 27K, 30K, 31K, 32K, 33K, and 36K.

In one embodiment, the GLP-1 analog comprises one or two Lys residues selected from the group consisting of: 12K, 21K, 23K, 24K, 25K, 26K, 27K, 30K, 31K, 32K , 33K, 34K and 36K.

In one embodiment, the GLP-1 analog comprises one or two Lys residues selected from the group consisting of: 21K, 23K, 24K, 25K, 26K, 27K, 30K, 31K, 32K, 33K And 34K.

In one embodiment, the GLP-1 analog comprises exactly two Lys residues selected from the group consisting of: 12K, 21K, 23K, 24K, 25K, 26K, 27K, 30K, 31K, 32K, 33K, 34K and 36K.

In one embodiment, the GLP-1 analog comprises exactly two Lys residues selected from the group consisting of: 21K, 23K, 24K, 25K, 26K, 27K, 30K, 31K, 32K, 33K and 34K.

In one embodiment, the GLP-1 analog comprises exactly two Lys residues selected from the following pairs:

a) 21K and 26K

b) 23K and 26K

c) 24K and 26K

d) 25K and 26K

e) 27K and 26K

f) 30K and 26K

g) 31K and 26K

h) 32K and 26K

i) 33K and 26K

j) 34K and 26K

In one embodiment, the GLP-1 analog comprises Lys residues 26K and 34K.

In one embodiment, the GLP-1 analog comprises exactly one Lys residue selected from 12K, 21K, 23K, 24K, 25K, 26K, 27K, 30K, 31K, 32K, 33K, 34K, and 36K.

In one embodiment, the GLP-1 analog comprises exactly one Lys residue selected from 21K, 23K, 24K, 25K, 26K, 27K, 30K, 31K, 32K, 33K, and 34K.

In one embodiment, the GLP-1 analog comprises exactly one Lys residue selected from 21K, 23K, 24K, 25K, 26K 27K 30K.

In one embodiment, the GLP-1 analog comprises exactly one 26K Lys residue.

In one embodiment, the GLP-1 analog comprises a substitution or deletion of one or both of 26K and 34K. In one embodiment, the GLP-1 analog does not contain 26K. In one embodiment, the GLP-1 analog comprises a deletion of 26K. In one embodiment, the GLP-1 analog comprises a 26K amino acid substitution. In one embodiment, the GLP-1 analog comprises 26R.

In one embodiment, the GLP-1 analog does not include 34K. In one embodiment, the GLP-1 analog comprises a deletion of 34K. In one embodiment, the GLP-1 analog comprises a 34K amino acid substitution. In one embodiment, the GLP-1 analog comprises 34R or 34Q.

As mentioned above, GLP-1 analogs according to the invention are similar in length to wt GLP-1. In one embodiment, the GLP-1 analog comprises at least 26 (such as at least 28 or at least 30) amino acid residues. In one embodiment, the GLP-1 analog comprises at least 31 (such as at least 32 or at least 33) amino acid residues

In one embodiment, the GLP-1 analogue has a deletion of 1-5 amino acids at the C-terminus. In one embodiment, the GLP-1 analog comprises a deletion of AA 35-37, AA 34-37, or AA 33-37.

In one embodiment, the GLP-1 analog comprises 33L.

As mentioned above, the GLP-1 analogue may comprise one or more amino acid substitutions, such as up to 5 amino acid substitutions, such as up to 4 amino acid substitutions, such as up to 3 amino acids substituted.

In one embodiment, the GLP-1 analogue has at least 75% identity with SEQ ID NO .: 127, such as 80%, such as 85, such as 90, or even 95% identity, in non-truncated examples Corresponding to up to 7, 6, 4, 4, 3 and 1 amino acid substitutions compared to SEQ ID NO 1, respectively.

The GLP-1 analog may contain one or more different amino acid residues in addition to or in addition to additional Lys residues introduced by amino acid substitution. The amino group is substituted by the amino group of the wt residue.

In one embodiment, the GLP-1 analog comprises an amino acid substitution of 8A, such as a substitution of 8A to 8G or 8W, which may also be referred to as A8G and A8W.

In one embodiment, the GLP-1 analog comprises an amino acid substitution of 8A, such as a substitution of 8A to a non-proteinogenic amino acid residue (such as Aib).

In one embodiment, the GLP-1 analog comprises an amino acid substitution of 8A to G, W or a non-proteinogenic amino acid residue Aib.

In one embodiment, the GLP-1 analog comprises one or more selected from positions 8, 12, 21, 23, 24, 25, 26, 27, 29, 30, 31, 32, 33, 34, and 36 Substituted with amino acids.

In one embodiment, the GLP-1 analog comprises one or more amino acids selected from positions 8, 21, 23, 24, 25, 26, 27, 29, 30, 31, 32, 33, and 34 Substituted amino acids.

In one embodiment, the GLP-1 analog comprises one or more amino-substituted amino groups selected from position 8, 21, 23, 24, 25, 27, 29, 30, 31, 32, or 33 Acid substitution. In one embodiment, the wt amino acid residues at positions 8, 21, 23, 24, 25, 27, 29, 30, 31, 32, or 33 are residues of G, V, A, T, L, or I Radical substitution.

In one embodiment, the GLP-1 analog comprises 8A and 34K substitutions.

In one embodiment, the GLP-1 analog comprises 8Aib and 34R. In one embodiment, the GLP-1 analog comprises 8Aib and 34R and a substitution at a position selected from positions 21, 23, 24, 25, 27, 29, 30, 31, 32, and 33.

In one embodiment, the GLP-1 analog comprises 8Aib and 34R. In one embodiment, the GLP-1 analog comprises 8Aib and 34R and a substitution at a position selected from positions 21, 23, 24, 25, 27, 29, 30, 31, 32, and 33, wherein positions 21, The substitutions of 23, 24, 25, 27, 29, 30, 31, 32 or 33 are G, V, A, T, L or I residues.

In one embodiment, the GLP-1 analog comprises one or two Lys residues and a selected group of substitutions:

In one embodiment, the GLP-1 analog comprises one or two Lys residues and a group selected from the group consisting of:

In one embodiment, such GLP-1 analogs comprise one or two Lys residues as described herein above, such as 26K and / or 34K, or such as 26K and / or Lys introduced by substitution with K34R .

In one embodiment, the GLP-1 analogue has a sequence consisting of SEQ ID NO 187: HX ₈ -EGTX ₁₂ -TSDVSSYLX ₂₁ -GX ₂₃ -X ₂₄ -X ₂₅ -X ₂₆ -X ₂₇ -FX ₂₉ -X ₃₀ -X ₃₁ -X ₃₂ -X ₃₃ -X ₃₄ -X ₃₅ -X ₃₆ -X ₃₇ , where X ₈ is A, G, W or Aib, X ₁₂ F or K

X ₂₁ series E, G or K

X ₂₃ series Q, G or K

X ₂₄ series A, G, V or K

X ₂₅ series A, G, V or K

X ₂₆ series K or R

X ₂₇ series E, G or K

X ₂₉ series I, A or V

X ₃₀ series A, G or K

X ₃₁ is W, G or K

X ₃₂ series L, G, T, V, I or K

X ₃₃ series V, G, I, L, K or absent

X ₃₄ is K, R, Q or does not exist

X ₃₅ series G or does not exist

X ₃₆ is R, K or does not exist

X ₃₇ is G or does not exist.

In one embodiment, the GLP-1 analogue has a sequence consisting of SEQ ID NO 187: HX ₈ -EGTX ₁₂ -TSDVSSYLX ₂₁ -GX ₂₃ -X ₂₄ -X ₂₅ -X ₂₆ -X ₂₇ -FX ₂₉ -X ₃₀ -X ₃₁ -X ₃₂ -X ₃₃ -X ₃₄ -X ₃₅ -X ₃₆ -X ₃₇ , where X ₈ is A, G, W or Aib, X ₁₂ F or K

X ₂₁ series E, G or K

X ₂₃ series Q, G or K

X ₂₄ series A, G, V or K

X ₂₅ series A, G, V or K

X ₂₆ series K or R

X ₂₇ series E, G or K

X ₂₉ series I or V

X ₃₀ series A, G or K

X ₃₁ is W, G or K

X ₃₂ series L, G, T, V, I or K

X ₃₃ series V, G, I, L, K or absent

X ₃₄ is K, R, Q or does not exist

X ₃₅ series G or does not exist

X ₃₆ is R, K or does not exist

X ₃₇ is G or does not exist.

In one embodiment, the GLP-1 analogue has a sequence consisting of SEQ ID NO 187: H-X ₈ -EGTX ₁₂ -TSDVSSYLX ₂₁ -GX ₂₃ -X ₂₄ -X ₂₅ -X ₂₆ -X ₂₇ -FX ₂₉ -X ₃₀ -X ₃₁ -X ₃₂ -X ₃₃ -X ₃₄ -X ₃₅ -X ₃₆ -X ₃₇ , where X ₈ is A, G, W or Aib, X ₁₂ F or K

X ₂₁ series E, G or K

X ₂₃ series Q, G or K

X ₂₄ series A, G, V or K

X ₂₅ series A, G, V or K

X ₂₆ series K or R

X ₂₇ series E, G or K

X ₂₉ series I, A or V

X ₃₀ series A, G or K

X ₃₁ is W, G or K

X ₃₂ series L, T, V, I or K

X ₃₃ series V, G, I, L, K or absent

X ₃₄ is K, R, Q or does not exist

X ₃₅ series G or does not exist

X ₃₆ is R, K or does not exist

X ₃₇ is G or does not exist.

In one embodiment, the GLP-1 analogue has a sequence consisting of SEQ ID NO 187: HX ₈ -EGTX ₁₂ -TSDVSSYLX ₂₁ -GX ₂₃ -X ₂₄ -X ₂₅ -X ₂₆ -X ₂₇ -FX ₂₉ -X ₃₀ -X ₃₁ -X ₃₂ -X ₃₃ -X ₃₄ -X ₃₅ -X ₃₆ -X ₃₇ , where X ₈ is A, G, W or Aib, X ₁₂ F or K

X ₂₁ series E, G or K

X ₂₃ series Q, G or K

X ₂₄ series A, G, V or K

X ₂₅ series A, G, V or K

X ₂₆ series K or R

X ₂₇ series E, G or K

X ₂₉ series I or V

X ₃₀ series A, G or K

X ₃₁ is W, G or K

X ₃₂ series L, V, I or K

X ₃₃ series V, G, I, L, K or absent

X ₃₄ is K, R, Q or does not exist

X ₃₅ series G or does not exist

X ₃₆ is R, K or does not exist

X ₃₇ is G or does not exist.

As can be seen, the examples herein include more than 40 GLP-1 analogs, which are also previously foreseen in compounds containing both GLP-1 analogs and EGF (A) analogs. These analogs are identified in the table below, in which the amino acid changes (as described above herein) compared to the wt residues are related to one or more Lys residues present in the analog Base is displayed together.

In one embodiment, the GLP-1 analog comprises or consists of an analog selected from the group of analogs defined by SEQ ID NO .: 138-186.

In one embodiment, the GLP-1 analog comprises or consists of an analog selected from the group of analogs defined by SEQ ID NO .: 138-142 and 144-186.

In one embodiment, the GLP-1 analog comprises or consists of the following: selected from the group of analogs defined by SEQ ID NO .: 138-142 and 144-166, 168, 169-186 Analogs.

In one embodiment, the GLP-1 analog comprises or consists of the following: selected from the group consisting of the similarities defined by SEQ ID NO .: 138-142 and 144-161, 163-166, 168, 169-186 An analog of a group of things.

In one embodiment, the GLP-1 analog comprises or consists of the following: selected from the group consisting of analogs defined by SEQ ID NO .: 138-142 and 145-161, 163-166, 168, 169-186 An analog of a group of things.

In one embodiment, the GLP-1 analog comprises or consists of an analog selected from the group of analogs defined by SEQ ID NO.:139 and 147-154.

In one embodiment, the GLP-1 analog comprises or consists of an analog selected from the group of analogs defined by SEQ ID NO .: 138 and 174-182 and 184-186.

In one embodiment, the GLP-1 analog comprises or consists of an analog selected from the group of analogs defined by SEQ ID NO.:166-170.

In one embodiment, the GLP-1 analog comprises or consists of an analog selected from the group of analogs defined by SEQ ID NO.:163-166.

In one embodiment, the GLP-1 analog comprises or consists of the following: an analog defined by SEQ ID NO.:164.

EGF (A) analog

The term "EGF (A) analog" refers herein to a variant of the EGF (A) domain (293-332) (SEQ ID NO: 1) of LDL-R. A nomenclature similar to that described above for the GLP-1 analogs is used for such EGF (A) analogs.

The terms "EGF (A) domain of LDL-R", "LDL-R (293-332)", "Native LDL-R (293-332)," EGF (A) (293-332) "," wild type "EGF (A)", "wt-EGF (A)" or "Native EGF (A)" is used herein to refer to the peptide consisting of the sequence SEQ ID NO: 1.

SEQ ID NO: Line 1: Gly-Thr-Asn-Glu-Cys-Leu-Asp-Asn-Asn-Gly-Gly-Cys-Ser-His-Val-Cys-Asn-Asp-Leu-Lys-Ile-Gly -Tyr-Glu-Cys-Leu-Cys-Pro-Asp-Gly-Phe-Gln-Leu-Val-Ala-Gln-Arg-Arg-Cys-Glu.

In this application, the amino acid residues are numbered according to the EGF (A) domain (LDL-R- (293-332)) for LDL-R, where the first (N-terminal) amino acid Residues are numbered or assigned position no.293, and subsequent amino acid residues towards the C-terminus are numbered 294, 295, 296, and so on, up to the final (C-terminal) amino acid residue, which In the EGF (A) domain of LDL-R is Glu with number 332.

Numbering is done in different ways in the sequence listing, where the first amino acid residue (Gly) of SEQ ID NO: 1 is designated as no.1, and the last amino acid residue (Glu) is designated as no.40. The same applies to other sequences in the Sequence Listing, that is, the designated N-terminal amino acid system no.1, regardless of the position of the reference LDL-R (293-332) relative to the 293Gly or 293 substituted amino acid residue . However, herein, the numbering of the amino acid positions is referenced to LDL-R (293-332), as explained above.

The level of agreement with SEQ ID NO .: 1 can be calculated by determining the number of amino acids that have not changed compared to SEQ ID NO 1. SEQ ID NO: 1 is composed of 40 amino acid residues, and if three amino acid substitutions are introduced, the level of consistency is 37/40% = 92.5%. If 5 amino acid residues are changed, the level of consistency is 87.5%. If the N- or C-terminus of the peptide is prolonged, this portion is usually not included in the comparison, but the absence of one or more amino acids shortens the comparator. For example, in the above example, if the N-terminal amino acid is deleted, the level of consistency is slightly reduced to 36 / 39X100% and 34 / 39X100%, respectively. When discussing the identity of the backbone sequence of a derivative, the amino acid residues of its substituents (eg, the residue to which the substituent is attached, also referred to as the amino acid residue of the substituent) may be wild (Wt) or substituted amino acids. If the amino acid residue of this substituent is a wild-type residue (such as 312K), this residue is included in the calculation of the level of consistency, but Lys at any other position from 293 to 332 will be amino acid substituted And it is not included when calculating the amino acid identity with SEQ ID NO.:1.

Each of the EGF (A) analogs of the present invention can be described by referring to: i) the natural EGF (A) (LDL-R (293-332)) corresponding to the amino acid residue being changed Numbering of amino acid residues (ie corresponding positions in natural LDL-R (293-332) EGF (A)), and ii) actual changes.

In other words, these EGF (A) analogs can be described by referring to natural LDL-R (293-332) EGF (A) peptides, that is, as some of the amino acid residues are equivalent to natural LDL-R (293- 332) A variant of EGF (A) (SEQ ID NO: 1) which has been changed by comparison. These changes may (independently) exhibit one or more amino acid substitutions.

The following are non-limiting examples of suitable analog nomenclature: EGF (A) analogs in Compound # 1, which are examples of derivatives comprising GLP-1 analogs and EGF (A) analogs, incorporated herein may be Called the following LDL-R (293-332) EGF (A) analogs: (301Leu, 309Arg, 312Glu, 321Glu) LDL-R (293-332) EGF (A), or (Leu301, Arg309, Glu312, Glu321) -LDL-R (293-332) EGF (A) or (301L, 309R, 312E, 321E) LDL-R (293-332) or (L301, R309, E312, E321) LDL-R (293-332). This means that when this analog is compared with natural LDL-R (293-332), it has i) located in the analog (according to the comparison) corresponding to natural LDL-R (293-332) EGF (A Leu at position 301 in), ii) Arg at position 309 corresponding to position 309 in natural LDL-R (293-332) EGF (A), iii) at position corresponding to this analog Glu, iv) of position 312 in natural LDL-R (293-332) EGF (A) is located in the analog corresponding to position 321 in natural LDL-R (293-332) EGF (A) Glu.

The expression "position equivalent to" or "corresponding position" can be used to define the variant LDL-R (293 by referring to the reference natural LDL-R (293-332) EGF (A) (SEQ ID NO: 1) -332) A characteristic of a changed position in the EGF (A) sequence.

Equivalent or corresponding positions (and the number of changes) are, for example, easily deduced by simple calculations and / or standard protein or peptide alignment programs such as "alignment" based on the Needleman-Wunsch algorithm can be used.

In one embodiment, the EGF (A) analog has 1-15 amino acid substitutions compared to SEQ ID NO.:1. In one embodiment, the EGF (A) analogue has 1-10 amino acid substitutions compared to SEQ ID NO.:1. In one embodiment, the EGF (A) analogue has 1-8 amino acid substitutions, such as 1-7, 1-6, 1-5 amino acid substitutions compared to SEQ ID NO.:1. In a particular embodiment, up to 7 amino acid substitutions may be present in the EGF (A) analog, for example, up to 6, 5, 4, 3, 2 or 1 amino acid substitutions may be present.

In one embodiment, the EGF (A) analogue is at least 75% identical to SEQ ID NO .: 1, such as 80%, such as 85, such as 90, or even 95% identical. In one embodiment in which no deletion / truncation is present, this corresponds to up to 10, 8, 6, 4, and 2 amino acid substitutions, respectively, compared to SEQ ID NO.

In one embodiment, the EGF (A) analogue is at least 90% identical to SEQ ID NO .: 1, such as 92%, such as 94, such as 96, or even 98% identical

In one embodiment, the EGF (A) analog comprises at least 35 (such as 36, 37, 38, 39, or at least 40) amino acids. In a particular embodiment, the EGF (A) analogue consists of 36 (such as 38 or 40) amino acids. In another particular embodiment, the EGF (A) analogue is composed of 35, 36, 37, 38, 39 or 40 amino acids.

If an amino acid addition is present (referred to herein as the N- and C-terminal extensions), the EGF (A) analog may contain up to 60 amino acids. In a particular embodiment, the EGF (A) analog comprises 35-60, 38-55, 40-50, 40-45, 40-42 or 40-41 amino acids. In one embodiment, the EGF (A) analogue is composed of 40 or 41 amino acid residues.

In one embodiment, the EGF (A) analog comprises an amino acid substitution of amino acid residue 301 from Asn to Leu, which is also described by Asn301Leu or 301Leu alone. In a particular embodiment, the EGF (A) analogue comprises the substitution 301Leu.

Additionally or alternatively, the EGF (A) analog comprises amino acid residues 297Cys, 304Cys, 308Cys, 317Cys, 319Cys, and 331Cys. These Cys residues are wild-type residues that can be linked by disulfide bridges, such as the disulfide bridges between 297Cys and 308Cys, 304Cys and 317Cys, and 319Cys and 331Cys.

In one embodiment, the EGF (A) analog comprises 301 Leu and some further amino acid substitutions, as described below.

In one embodiment, the EGF (A) analog comprises amino acid substitutions of 301Leu, 310Asp, and 312Lys.

In one embodiment, the EGF (A) analog comprises 301Leu and 310Asp and wherein the analog does not have a substitution of 299Asp to Glu, VaL or His.

In one embodiment, the EGF (A) analog comprises 301Leu, 309Arg and 312Glu.

In one embodiment, the EGF (A) analog comprises 301Leu, 309Arg, 312Glu, and 321Glu.

In one embodiment, the EGF (A) analogue includes 301Leu and 309Arg, a prerequisite is that the analogue does not have a substitution of 310Asp to 310Lys or in one embodiment, the EGF (A) analogue includes 301Leu and 309Arg The prerequisite is that the analog does not have a substitution of 299Asp to Glu, VaL or His.

In a further embodiment, the EGF (A) analog does not have any substitution of D310K, D310N, D310Q, D310Q, D310R, and D310A or even any 310Asp.

In one embodiment, the EGF (A) analog comprises one, two, three, or all four of the following wild-type residues: 295Asn, 296Glu, 298Leu, and 302Gly.

In one embodiment, the EGF (A) analog comprises one, two, three, four, or all five of the following wild-type residues: 295Asn, 296Glu, 298Leu, 302Gly, and 310Asp.

In one embodiment, the peptide has 295Asn.

In one embodiment, the EGF (A) analog has 296Glu. In one embodiment, the EGF (A) analog has 298Leu. In one embodiment, the EGF (A) analog has 302 Gly. In one embodiment, the EGF (A) analog has 310 Asp.

In one embodiment, the EGF (A) analog has two or more of 310 Asp, 295 Asn, and 296 Glu. In one embodiment, the EGF (A) analog has all three of 310Asp, 295Asn, and 296Glu.

The EGF (A) analog may comprise further amino acid substitutions as described herein. In one embodiment, the analog may further comprise one or more amino acid substitutions at a position selected from the group consisting of: 293, 294, 296, 299, 300, 303, 305, 306, 309, 311, 312, 313, 314, 315, 316, 318, 320, 321, 322, 323, 324, 325, 326, 328, 329, 330 and 332.

In one embodiment, the analog may further comprise one or more amino acid substitutions at a position selected from the group consisting of: 293, 294, 299, 300, 303, 305, 306, 309, 311, 312, 313, 314, 316, 318, 321, 322, 323, 324, 325, 326, 328, 329, 330, 331 and 332.

In one embodiment, the analog may further comprise one or more amino acid substitutions at a position selected from the group consisting of 294, 299, 300, 303, 309, 312, 313, 314, 316, 318, 321 , 322, 323, 324, 325, 326, 328, 329, 330, and 332.

In one embodiment, the analog may further comprise one or more amino acid substitutions at a position selected from the group consisting of 299, 300, 309, 313, 316, 318, 321, 322, 323, 324, 326 , 328, 329, 330, and 332.

In one embodiment, the analog may further comprise one or more amino acid substitutions at positions selected from the group consisting of: 309, 312, 313, 321, 324, 328, and 332.

In a further embodiment, in addition to the amino acid residues specified above herein, the EGF (A) analogue contains wt amino acid residues or different residues (i.e., amino groups) at certain special positions. Acid substitution).

In one such embodiment, the EGF (A) analog comprises the amino acid residue Gly (G) or Asn (N) at position 293.

In one such embodiment, the EGF (A) analog comprises the amino acid residue Trp (W), Thr (T), or Gly (G) at position 294.

In one such embodiment, the EGF (A) analog comprises the amino acid residues Asp (D), Gly (G), Pro (P), Arg (R), Lys (K), Ser at position 299 (S), Thr (T), Asn (N), Gln (Q), Ala (A), Ile (I), Leu (L), Met (M), Phe (F), Tyr (Y) or Trp (W).

In one such embodiment, the EGF (A) analog comprises the amino acid residues Asp (D), Gly (G), Pro (P), Arg (R), Lys (K), Ser at position 299 (S), Thr (T), Asn (N), Gln (Q), Ala (A), Met (M), Phe (F), Tyr (Y), or Trp (W).

In one such embodiment, the EGF (A) analog comprises the amino acid residues Asp (D), Ser (S), Arg (R), Leu (L), Ala (A), Lys at position 299 (K) or Tyr (Y).

In one such embodiment, the EGF (A) analog comprises the amino acid residue Asp (D) or Ala (A) at position 299.

In one such embodiment, the EGF (A) analog comprises an amino acid residue His (H) or Asn (N) at position 300.

In one such embodiment, the EGF (A) analog comprises an amino acid residue Val (V), Ser (S), Thr (T), or Ile (I) at position 307.

In one such embodiment, the EGF (A) analog comprises the amino acid residue Val (V) or Ile (I) at position 307.

In one such embodiment, the EGF (A) analog comprises Ser (S), Thr (T), or Ile (I) at position 307.

In one such embodiment, the EGF (A) analog comprises Ile (I) at position 307.

In one such embodiment, the EGF (A) analog comprises the amino acid residue Asn (N), Glu (E), His (H,) Arg (R), Ser (S), or Lys at position 309 (K).

In one such embodiment, the EGF (A) analog of the present invention comprises an amino acid residue Asn (N), Arg (R), Ser (S), or Lys (K) at position 309.

In one such embodiment, the EGF (A) analog comprises the amino acid residue Asn (N), Arg (R), or Ser (S) at position 309.

In one such embodiment, the EGF (A) analog comprises the amino acid residue Asn (N) or Arg (R) at position 309.

In one such embodiment, the EGF (A) analog comprises the amino acid residue Lys (K) or Arg (R) at position 309.

The EGF (A) analog may comprise several amino acid substitutions as described herein, such as one or more amino acid substitutions selected from the group consisting of 299Ala, 307Ile, and 321Glu.

In a further embodiment, the EGF (A) analog comprises the amino acid residue Asp (D), Lys (K) or Glu (E) at position 321.

In a further embodiment, the EGF (A) analog comprises the amino acid residue Asp (D) or Glu (E) at position 321.

In a further embodiment, the EGF (A) analog comprises the amino acid residue Glu (E) at position 321.

In a further embodiment, the EGF (A) analog comprises the amino acid residue Gln (Q) or Gly (G) at position 324.

In a further embodiment, the EGF (A) analog comprises the amino acid residue Arg (R) or His (H) at position 329.

In a further embodiment, the EGF (A) analog does not have a substitution of 300Asn (N) for Pro (P).

The EGF (A) domain of LDL-R includes an lysine at position 312, which can be used for substitution as described herein. In embodiments in which the attachment of substituents to 312 is not desired, 312Lys may be substituted with another amino acid, as described herein.

In one embodiment, the EGF (A) analog does not contain Lys residues.

In one embodiment, Lys at position 312 is substituted with an amino acid residue selected from the group consisting of Gly, Pro, Asp, Glu, Arg, His, Ser, Thr, Asn, Gln, Ala, Val, Ile , Leu, Met, Phe, and Tyr. In one embodiment, Lys at position 312 is substituted with an amino acid residue selected from the group consisting of Gly, Asp, Glu, Ser, Thr, Asn, Ala, Val, Ile, Leu, Phe, and Tyr. In one embodiment, Lys at position 312 is substituted with an amino acid residue selected from the group consisting of Asp, Glu, Thr, Asn, Ile, Leu, Phe, and Tyr. In one embodiment, 312Lys is substituted with 312Asp, 312Glu, 312Thr, 312Asn, 312Ile, or 312Phe. In one embodiment, 312Lys is substituted with 312Glu, 312Asp, 312Gln or 312Arg.

In one embodiment, 312Lys is substituted with 312Glu, 312Thr, 312Asn, 312Ile, 312Phe, or 312Tyr. In one embodiment, 312Lys is replaced by 312Glu, 312Asn or 312Ile,

In one embodiment, 312Lys is substituted with 312Glu or 312Arg. In one embodiment, 312Lys is substituted with 312Arg. In one embodiment, 312Lys is substituted with 312Glu.

To include options for substituents attached to various positions (see further below), Lys may be substituted by the amino acid of the wild-type residue of SEQ ID NO .: 1 or by SEQ ID NO .: 1 of Peptide extension (such as 292Lys or a 333Lys) is introduced.

In examples where more than one substituent is desired, one may be via 312Lys and the second via Lys introduced by extension or substitution of the peptide in SEQ ID NO.:1.

In one embodiment, the EGF (A) analog of SEQ ID NO: 1 comprises at least one Lys residue at a position selected from the group consisting of: 292Lys, 293Lys, 294Lys, 296Lys, 299Lys, 300Lys, 303Lys, 305Lys, 306Lys, 309Lys, 311Lys, 312Lys, 313Lys, 314Lys, 315Lys, 316Lys, 318Lys, 320Lys, 321Lys, 322Lys, 323Lys, 324Lys, 325Lys, 326Lys, 327Lys, 328Lys, 329Lys, 330Lys, 332Lys, and 333Lys.

In one embodiment, the EGF (A) analog of SEQ ID NO: 1 comprises at least one Lys residue at a position selected from the group consisting of: 292Lys, 293Lys, 294Lys, 299Lys, 300Lys, 303Lys, 305Lys, 306Lys, 309Lys, 311Lys, 312Lys, 313Lys, 314Lys, 315Lys, 316Lys, 318Lys, 320Lys, 321Lys, 322Lys, 323Lys, 324Lys, 325Lys, 326Lys, 327Lys, 328Lys, 329Lys, 330Lys, 332Lys and 333Lys.

In one embodiment, the EGF (A) analog of SEQ ID NO: 1 comprises at least one Lys residue at a position selected from the group consisting of: 292Lys, 293Lys, 294Lys, 300Lys, 303Lys, 305Lys, 306Lys, 309Lys, 311Lys, 312Lys, 313Lys, 314Lys, 316Lys, 318Lys, 321Lys, 322Lys, 323Lys, 324Lys, 325Lys, 326Lys, 327Lys, 328Lys, 329Lys, 330Lys, 332Lys, and 333Lys.

In one embodiment, the EGF (A) analog of SEQ ID NO: 1 comprises at least one Lys residue at a position selected from the group consisting of: 292Lys, 293Lys, 294Lys, 300Lys, 303Lys, 305Lys, 306Lys, 311Lys, 312Lys, 313Lys, 314Lys, 316Lys, 318Lys, 322Lys, 323Lys, 324Lys, 325Lys, 326Lys, 327Lys, 328Lys, 329Lys, 330Lys, 332Lys and 333Lys.

In one embodiment, the EGF (A) analog of SEQ ID NO: 1 comprises at least one Lys residue at a position selected from the group consisting of: 292Lys, 293Lys, 294Lys, 300Lys, 303Lys, 305Lys, 306Lys, 311Lys, 313Lys, 314Lys, 316Lys, 318Lys, 322Lys, 323Lys, 324Lys, 325Lys, 326Lys, 327Lys, 328Lys, 329Lys, 330Lys, 332Lys, and 333Lys.

Additionally or alternatively, the EGF (A) analog of the present invention comprises at least one amino acid substitution selected from the group consisting of 292Lys, 293Lys, 294Lys, 295Lys, 296Lys, 298Lys, 299Lys, 301Lys, 302Lys, 303Lys, 305Lys, 306Lys, 307Lys, 309Lys, 310Lys, 311Lys, 313Lys, 314Lys, 315Lys, 316Lys, 318Lys, 320Lys, 321Lys, 322Lys, 323Lys, 324Lys, 325Lys, 326Lys, 327Lys, 328Lys, 329Lys, 330Lys, 332Lys, and 333Lys.

In a further embodiment, the EGF (A) analog of the present invention comprises at least one amino acid substitution selected from the group consisting of: 292Lys, 293Lys, 294Lys, 295Lys, 296Lys, 298Lys, 299Lys, 302Lys, 303Lys, 305Lys, 306Lys , 307Lys, 309Lys, 311Lys, 313Lys, 314Lys, 315Lys, 316Lys, 318Lys, 320Lys, 321Lys, 322Lys, 323Lys, 324Lys, 325Lys, 326Lys, 327Lys, 328Lys, 329Lys, 330Lys, 332Lys and 333Lys.

In a further embodiment, the EGF (A) analog of the present invention comprises at least one amino acid substitution selected from the group consisting of: 292Lys, 293Lys, 294Lys, 295Lys, 296Lys, 298Lys, 299Lys, 303Lys, 305Lys, 306Lys, 309Lys , 311Lys, 313Lys, 314Lys, 315Lys, 316Lys, 318Lys, 320Lys, 321Lys, 322Lys, 323Lys, 324Lys, 325Lys, 326Lys, 327Lys, 328Lys, 329Lys, 330Lys, 332Lys and 333Lys.

In a further embodiment, the EGF (A) analog of the present invention comprises at least one amino acid substitution selected from the group consisting of: 292Lys, 293Lys, 294Lys, 295Lys, 296Lys, 299Lys, 303Lys, 305Lys, 306Lys, 309Lys, 311Lys , 313Lys, 314Lys, 315Lys, 316Lys, 318Lys, 320Lys, 321Lys, 322Lys, 323Lys, 324Lys, 325Lys, 326Lys, 327Lys, 328Lys, 329Lys, 330Lys, 332Lys, and 333Lys.

In a further embodiment, the EGF (A) analog peptide of the present invention comprises at least one amino acid substitution selected from the group consisting of: 292Lys, 293Lys, 294Lys, 296Lys, 299Lys, 303Lys, 305Lys, 306Lys, 309Lys, 311Lys , 313Lys, 314Lys, 315Lys, 316Lys, 318Lys, 320Lys, 321Lys, 322Lys, 323Lys, 324Lys, 325Lys, 326Lys, 327Lys, 328Lys, 329Lys, 330Lys, 332Lys, and 333Lys.

In a further embodiment, the EGF (A) analog of the present invention comprises at least one amino acid substitution selected from the group consisting of: 292Lys, 293Lys, 294Lys, 299Lys, 303Lys, 305Lys, 306Lys, 309Lys, 311Lys, 313Lys, 314Lys , 315Lys, 316Lys, 318Lys, 320Lys, 321Lys, 322Lys, 323Lys, 324Lys, 325Lys, 326Lys, 327Lys, 328Lys, 329Lys, 330Lys, 332Lys, and 333Lys.

In a further embodiment, the EGF (A) analog of the present invention comprises at least one amino acid substitution selected from the group consisting of: 292Lys, 293Lys, 294Lys, 299Lys, 303Lys, 305Lys, 306Lys, 309Lys, 311Lys, 313Lys, 314Lys , 315Lys, 316Lys, 318Lys, 320Lys, 321Lys, 322Lys, 323Lys, 324Lys, 325Lys, 326Lys, 327Lys, 328Lys, 329Lys, 330Lys, 332Lys, and 333Lys.

In a further embodiment, the EGF (A) analog of the present invention comprises at least one amino acid substitution selected from the group consisting of: 292Lys, 293Lys, 294Lys, 299Lys, 303Lys, 305Lys, 306Lys, 310Lys, 311Lys, 313Lys, 314Lys , 315Lys, 316Lys, 318Lys, 320Lys, 321Lys, 322Lys, 323Lys, 324Lys, 325Lys, 326Lys, 327Lys, 328Lys, 329Lys, 330Lys, 332Lys, and 333Lys.

In a further embodiment, the EGF (A) analog of the present invention comprises at least one amino acid substitution selected from the group consisting of: 292Lys, 293Lys, 294Lys, 299Lys, 303Lys, 305Lys, 306Lys, 309Lys, 310Lys, 311Lys, 313Lys , 314Lys, 315Lys, 316Lys, 318Lys, 321Lys, 322Lys, 323Lys, 324Lys, 325Lys, 326Lys, 327Lys, 328Lys, 329Lys, 330Lys, 332Lys, and 333Lys.

In a further embodiment, the EGF (A) analog of the present invention comprises at least one amino acid substitution selected from the group consisting of: 292Lys, 293Lys, 294Lys, 303Lys, 305Lys, 306Lys, 310Lys, 311Lys, 313Lys, 314Lys, 315Lys , 316Lys, 318Lys, 321Lys, 322Lys, 323Lys, 324Lys, 325Lys, 326Lys, 327Lys, 328Lys, 329Lys, 330Lys, 332Lys, and 333Lys. In one embodiment, the EGF (A) analog of the present invention does not include any of the following substitutions: 296K, 298K, 301K, 302K, and 307K.

In one embodiment, the EGF (A) analog comprises any of the following substitutions: 296K, 298K, 301K, 302K, 307K, and 310K.

In one embodiment, the EGF (A) analog comprises any of the following substitutions: 296K, 298K, 301K, 302K, 307K, and 295K.

In one embodiment, the EGF (A) analog comprises any of the following substitutions: 296K, 298K, 301K, 302K, 307K, and 295D.

In a particular embodiment, the EGF (A) analog comprises one or two of such Lys substitutions.

Additionally or alternatively, the EGF (A) analog may comprise 312Lys.

In one embodiment, the EGF (A) analog of the present invention comprises two Lys residues. In one embodiment, the EGF (A) analog of the present invention comprises two paired Lys residues selected from the group consisting of:

In a further embodiment, the EGF (A) analog compared to SEQ ID NO .: 1 comprises at least two amino acid substitutions recognized by any of Groups I-XXIV shown below.

In a still further embodiment, the EGF (A) analog is composed of an amino acid substitution recognized by any of Groups I-XXIV as shown below.

In a further embodiment, the EGF (A) analog compared to SEQ ID NO .: 1 comprises at least two amino acid substitutions recognized by any of Groups I-XXIV shown below.

In a still further embodiment, the EGF (A) analog is composed of an amino acid substitution recognized by any of Groups I-XXIV as shown below.

I. 301Leu and 309Arg

II. 301Leu, 309Arg, 312Glu

III. 301Leu, 307Ile, and 309Arg

IV. 301Leu, 307Ile, 309Arg and 312Glu

V. 301Leu, 309Arg and 321Glu

VI. 301Leu, 309Arg, 321Glu and 312Glu

VII. 301Leu, 307Ile, 309Arg and 299Ala

VIII. 301Leu, 307Ile, 309Arg, 299Ala, and 312Glu

IX. 301Leu and 309Arg and at least one Lys substitution

X. 301Leu, 309Arg, 312Glu and at least one Lys substitution

XI. 301Leu, 307Ile, and 309Arg and at least one Lys substitution

XII. 301Leu, 307Ile, 309Arg and 312Glu and at least one Lys substitution

XIII. 301Leu, 309Arg and 321Glu and at least one Lys substitution

XIV. 301Leu, 309Arg, 321Glu and 312Glu and at least one Lys substitution

XV. 301Leu, 307Ile, 309Arg and 299Ala and at least one Lys substitution or XVI. 301Leu, 307Ile, 309Arg, 299Ala and 312Glu and at least one Lys substitution.

In a further embodiment, the EGF (A) analogue comprises at least two amino acid substitutions identified by any of the groups XVII-XX shown below compared to SEQ ID NO.:1.

In yet a further embodiment, the EGF (A) analog, compared to SEQ ID NO.:1, consists of an amino acid substitution recognized by any of the groups XVII-XX as shown below.

XVII. 301Leu and 309Lys

XVIII. 301Leu, 309Lys, and 312Glu

XIX. 301Leu and 309Lys and at least one further Lys substitution

XX. 301Leu, 309Lys and 312Glu and at least one further Lys substitution.

In a further embodiment, the EGF (A) analogue according to the present invention, compared to SEQ ID NO.:1, comprises at least two amino acid substitutions recognized by any of the groups XXI-XXIV shown below.

In a still further embodiment, the EGF (A) analog of the present invention is compared to SEQ ID NO .: 1 is composed of an amino acid substitution recognized by any of the groups XXI-XXIV as shown below .

XXI. 301Leu and 307Ile, XXII. 301Leu, 307Ile and 312Glu XXIII. 301Leu and 307Ile and at least one further Lys substitution and XXIV. 301Leu and 3307Ile and 312Glu and at least one further Lys substitution.

In a further specific embodiment, the EGF (A) analog comprises or consists of any one of the amino acid sequences identified by SEQ IDs 1 to 114.

In one embodiment, the EGF (A) analog comprises or consists of any one of the amino acid sequences identified by SEQ ID NO .: 2-114.

In one embodiment, the EGF (A) analog comprises or consists of any one of the amino acid sequences identified by SEQ ID NO .: 2-47 and 49-114.

In one embodiment, the EGF (A) analog comprises or consists of the following: identified by any of the amino acid sequences SEQ ID NO .: 2-44, 46, 47, and 49-114 Recognized amino acid sequence.

In one embodiment, the EGF (A) analog comprises or consists of the following: identified by SEQ ID NO .: 2-44, 46, 47, 49-53, 55, 58-1145 Any of the amino acid sequences.

In one embodiment, the EGF (A) analog comprises or consists of the following: consisting of SEQ ID NO .: 2-4, 6-44, 46, 47, 49-53, 55, 58- Any of the 114 identified amino acid sequences.

In one embodiment, the EGF (A) analog comprises or consists of the following: consisting of SEQ ID NO .: 2-4, 6-19, 21-44, 46, 47, 49-53, 55, 58-114 Any of the amino acid sequences recognized.

In one embodiment, the EGF (A) analog comprises or consists of the following: SEQ ID NO .: 19, 21, 73, 107, 108, 109, 110, 111, 112, 113, Any of the 114 identified amino acid sequences.

In one embodiment, the EGF (A) analog as described above does not contain Lys residues and the EGF (A) analog therefore comprises or consists of the following: consisting of SEQ ID NO .: 5, 6 , 23, 26, 49, 50, 107-111 of any of the amino acid sequences recognized.

In one embodiment, the EGF (A) analog comprises or consists of the amino acid sequence identified by SEQ ID NO .: 5, 6, 23, 26, 49, 50, or 107 Either.

In a preferred embodiment, the EGF (A) analog contains both 312K residues and 321D residue mutations and has no Lys residues, such as where the EGF (A) analog contains the following or is caused by Consisting of any one of the amino acid sequences identified by SEQ ID NO .: 108, 109, 110, or 111.

In one embodiment, the EGF (A) analog comprises or consists of the amino acid sequence identified by SEQ ID NO .: 108.

The examples herein provide a variety of EGF (A) analogs, which are included in the table below (including information on amino acid substitutions, Lys residues, and SEQ ID NO).

Fusion peptide

In one aspect, the invention relates to a fusion polypeptide comprising an amino acid sequence of a GLP-1 analog and an amino acid sequence of an EGF (A) analog. As previously described herein, analogs of GLP-1 refer to variants of (7-37) (SEQ ID No: 137) and analogs of EGF (A) refer to EGF (A of LDL-R) ) Domain (293-332) (SEQ ID NO: 1).

The fusion polypeptide may be further considered and intermediated in the preparation of derivatives as described herein below. When referring to a derivative of the invention, the fusion polypeptide may be referred to as the backbone or peptide backbone.

The preparation of fusion proteins or fusion polypeptides is widely known in the art. A recombinant vector for expressing the fusion polypeptide in a suitable host can be prepared and used to make a fusion protein based on general general knowledge by heterologous expression (Sambrook et al., Molecular Cloning: a laboratory manual, 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). Alternatively, shorter polypeptides are often manufactured by solid-phase peptide synthesis and even longer-length peptides can be synthetically manufactured. Peptide elements can also be manufactured separately and subsequently subjected to natural chemical ligation to produce a complete fusion polypeptide.

When two peptide segments are to be fused, their order can affect the functionality of the resulting fusion polypeptide and the compound containing it.

In one embodiment, according to the present invention, the order of the GLP-1 analogue and the EGF (A) analogue from the N-terminus is the GLP-1 analogue and then the EGF (A) analogue. Separated by spacer peptides (see below). It can be said that the GLP-1 analog is fused to the EGF (A) analog through the C-terminus of the GLP-1 analog.

In an alternative embodiment, the GLP-1 analog is placed at the N-terminus by fusing the C-terminus of the EGF (A) analog with the EGF (A) analog. The resulting fusion polypeptide can be referred to by the term "backbone" or "peptide backbone", which is defined to include both the GLP-1 analog and the EGF (A) analog, and if necessary, as described herein below. Spacer peptide peptides.

In one embodiment, the compounds, fusion polypeptides, and derivatives thereof of the present invention include GLP-1 analogs as defined herein above, including any of the analogs defined by SEQ ID NO.:138-187 By.

In one embodiment, the compounds, fusion polypeptides and derivatives thereof of the present invention comprise an EGF (A) analogue as defined herein above, which includes the analogues defined by SEQ ID NOs: 2-114 Anyone.

Spacer

Frequently, fusion polypeptides include spacers to ensure that any functionality present at the ends of two peptides is not disturbed by the proximity of the other peptide. In one embodiment, the spacer is a peptide, which is referred to herein as a spacer peptide or a peptide spacer. Various spacer peptides are known in the art and can be placed between the GLP-1 analog and the EGF (A) analog to obtain a fusion polypeptide. As described above, the resulting fusion polypeptide (including spacers) can be manufactured synthetically or by heterologous expression.

The spacer peptide is usually a peptide segment of 4-80 amino acids.

The following include examples of such peptides used herein.

In one embodiment, the compounds, fusion polypeptides, and derivatives thereof of the present invention comprise a peptide spacer, wherein the peptide spacer comprises a sequence selected from the peptides identified by SEQ ID NOs 115-136.

In one embodiment, the compounds, fusion polypeptides, and derivatives thereof of the present invention comprise a peptide spacer selected from the group of peptide spacers identified by SEQ ID NOs 115-136.

In one embodiment, the compounds, fusion polypeptides, and derivatives thereof of the present invention include peptide spacers, which include one or more GQAP segments, such as 1-20, such as 1-10, such as 1 -6, such as 1, 2, 3, 4, or 5 GQAP segments.

In one embodiment, the peptide spacer comprises a peptide spacer selected from the group of peptides identified by SEQ ID NO .: 115-128.

In one embodiment, the peptide spacer is selected from the sequence identified by SEQ ID NO .: 115-128.

In one embodiment, the peptide spacer does not include a Lys residue.

In one embodiment, the peptide spacer comprises a Lys residue.

In one embodiment, the compounds, fusion polypeptides, and derivatives thereof of the present invention comprise a peptide spacer comprising one or more GQAP segments, wherein the Lys residue is introduced by amino acid substitution. In a further such embodiment, the spacer may be selected from the group of sequences recognized by SEQ ID NO .: 121-128.

In one embodiment, the compounds, fusion polypeptides, and derivatives thereof of the present invention include peptide spacers that are glycine-rich, such as where at least half of its amino acid residues are Gly (such as amino acid residues) At least ¾ is a peptide spacer of Gly). In such an embodiment, the peptide spacer may be selected from the peptides identified by SEQ ID NO .: 130-136.

In a further embodiment, the peptide spacer is selected from the group of peptides identified by SEQ ID NOs: 115-117 and 121-136.

In a further embodiment, the peptide spacer is selected from the group of peptides identified by SEQ ID NOs: 115-117 and 121-128.

In one embodiment, the peptide spacer is selected from the sequence identified by SEQ ID NO .: 115-128.

In a further embodiment, the peptide spacer is selected from the group of peptides identified by SEQ ID NOs: 115-117. In a further embodiment, the peptide spacer is identified by SEQ ID NO: 116.

Multiple examples of the fusion polypeptide (peptide backbone) according to the invention are provided in the examples, which are shown in all elements (i.e. the GLP-1 analog, the EGF (A) analog and the peptide spacer Sub).

In one embodiment, the fusion polypeptide or backbone sequence of a derivative of the invention consists of a GLP-1 analog, an EGF (A) analog, and a peptide spacer as defined herein.

Examples of the present application include a plurality of such fusion polypeptides and derivatives including such fusion polypeptides as peptide backbones. Why the fusion polypeptide can be obtained from the sequence of individual elements (that is, the GLP-1 analog, the EGF (A) analog, and the peptide spacer, which together form a fusion polypeptide that individually specifies a SEQ ID according to the following table) infer.

Examples of GLP-1 analogs having the C-terminus of the EGF (A) analog

In one embodiment, the invention relates to a fusion polypeptide selected from the group of fusion polypeptides defined by: SEQ ID NO .: 188-384, 386-387.

In one embodiment, the invention relates to a fusion polypeptide selected from the group of fusion polypeptides defined by: SEQ ID NO .: 188-384.

In one embodiment, the present invention relates to a fusion polypeptide selected from the group of fusion polypeptides defined by: SEQ ID NO .: 193, 226-233, and 381-384.

In one embodiment, the invention relates to a fusion polypeptide selected from the group of fusion polypeptides defined by: SEQ ID NO .: 379-380.

In one embodiment, the invention relates to a fusion polypeptide selected from the group of fusion polypeptides defined by: SEQ ID NO .: 193, 219, and 220.

In one embodiment, the invention relates to a fusion polypeptide selected from the group of fusion polypeptides defined by: SEQ ID NO .: 189, 193, 200-203, and 212-218.

In one embodiment, the invention relates to a fusion polypeptide selected from the group of fusion polypeptides defined by: SEQ ID NO.:222-225.

In one embodiment, the invention relates to a fusion polypeptide selected from the group of fusion polypeptides defined by: SEQ ID NO .: 224-225.

In one embodiment, the invention relates to a fusion polypeptide selected from the group of fusion polypeptides defined by: SEQ ID NO .: 192-196.

In one embodiment, the invention relates to a fusion polypeptide selected from the group of fusion polypeptides defined by: SEQ ID NO .: 221, 236, 250, 264, 276, 279, 291, 294, 306, 307, 310, 324, 336, 339, 351, 352, 353, 356, 368 and 369.

In one embodiment, the invention relates to a fusion polypeptide selected from the group of fusion polypeptides defined by: SEQ ID NO .: 221, 250, 276, 279, 291, 294, 306, 307, 310, 324, 336, 351, 353, 356, 368 and 369.

In one embodiment, the invention relates to a fusion polypeptide selected from the group of fusion polypeptides defined by: SEQ ID NO .: 217, 218, 219, 220, 221, 310, and 386. In one embodiment, the invention relates to a fusion polypeptide selected from the group of fusion polypeptides defined by: SEQ ID NO .: 217, 218, 221, 310, and 386. In one embodiment, the invention relates to a fusion polypeptide selected from the group of fusion polypeptides defined by: SEQ ID NO .: 217, 218, 310, and 386. In one embodiment, the present invention relates to a fusion polypeptide selected from the group of fusion polypeptides defined by: SEQ ID NO .: 221, 310, and 386. In one embodiment, the invention relates to a fusion polypeptide selected from the group of fusion polypeptides defined by: SEQ ID NO .: 310 and 386. In one embodiment, the invention relates to a fusion polypeptide as defined by SEQ ID NO.:310.

In one embodiment, the invention relates to a fusion polypeptide selected from the group of fusion polypeptides defined by: SEQ ID NO .: 188 and 370-378.

In one embodiment, the invention relates to a fusion polypeptide selected from the group of fusion polypeptides defined by: SEQ ID NO .: 190, 191, 197, 198, and 199.

In one embodiment, the invention relates to a fusion polypeptide selected from the group of fusion polypeptides defined by: SEQ ID NO .: 204-211.

In one embodiment, the invention relates to a fusion polypeptide selected from the group of fusion polypeptides defined by: SEQ ID NO .: 240-247, 254-261, 268-275, 283-290, 298-305, 314-321, 328-335, 343-350, and 360-367.

In one embodiment, the invention relates to a fusion polypeptide selected from the group of fusion polypeptides defined by: SEQ ID NO .: 234-235, 237-239, 248-249, 251-253, 262-263, 265-267, 277-278, 280-282, 292-293, 295-297, 308-309, 311-313, 322-323, 325-327, 337-338, 340-342, 354-355 and 357- 359.

In one embodiment, the invention relates to a fusion polypeptide comprising exactly one Lys residue.

In one embodiment, the invention relates to a fusion polypeptide comprising up to two Lys residues.

In one embodiment, the invention relates to a fusion polypeptide comprising two Lys residues.

In one embodiment, the present invention pertains to a derivative comprising a fusion polypeptide or peptide backbone defined by SEQ ID NO .: 188-384, or any of the fusion polypeptides defined above, as described further below herein of.

GLP-1 function

Receptor agonists can be defined as analogs that bind to a receptor and elicit a response typically elicited by a natural ligand. Full agonists can be defined to elicit reacters on the same scale as natural ligands (see, eg, "Principles of Biochemistry", AL Lehninger, DL Nelson, MM Cox, Second Edition, Worth Publishers, 1993, p. 763).

Thus, for example, a "GLP-1 receptor agonist" can be defined as a compound capable of binding to the GLP-1 receptor and capable of activating it.

And a "complete" GLP-1 receptor agonist can be defined as a GLP-1 receptor agonist capable of eliciting a GLP-1 receptor response similar to that of natural GLP-1.

In one embodiment, the GLP-1 analogue of the present invention is a GLP-1 receptor agonist. In some embodiments, the GLP-1 analogs of the invention are full GLP-1 receptor agonists. In some embodiments, the bifunctional compound of the invention is a GLP-1 receptor agonist. In some embodiments, the bifunctional compound of the invention is a full GLP-1 receptor agonist. In some embodiments, the derivatives of the invention are GLP-1 receptor agonists. In some embodiments, the derivatives of the invention are full GLP-1 receptor agonists.

Therefore, the GLP-1 receptor agonist should show "GLP-1 activity", which refers to the binding of the compound (i.e., a GLP-1 analog or a compound containing a GLP-1 analog) to the GLP-1 receptor It also initiates the ability of a signaling pathway to cause insulinotropic effects or other physiological effects (as known in the art). For example, the GLP-1 analogs, bifunctional compounds, and derivatives thereof can be tested for GLP-1 activity using the GLP-1 potency assay described in the method section C. herein. In one embodiment, the GLP-1 analogs or compounds containing the GLP-1 analogs (i.e., the GLP-1 / EGF (A) fusion polypeptide and derivatives thereof) have GLP-1 activity .

The term half maximal effective concentration (EC ₅₀₎ induced at a concentration generally based addressed (by reference to a dose response curve) of the reaction intermediate and maximum baseline. EC ₅₀ is used as a measure of the potency of a compound and represents the observed concentration of 50% of its maximum potency.

The in-vitro potency of the GLP-1 analogs and compounds containing the GLP-1 analogs can be determined as described above, and the EC ₅₀ determination is performed. The lower the EC ₅₀ value, the better the effectiveness.

In one embodiment, the GLP-1 analog or a compound comprising such GLP-1 analogs has up to 50 pM, 50-100 pM, 100 in a GLP-1 in-vitro potency analysis (without HSA) described in C1 EC50 of -250pM or 250-1000pM. In one embodiment, the EC50 is at most 500 pM, such as at most 300 pM, such as at most 200 pM. In one embodiment, the EC50 is comparable to human GLP-1 (7-37), such as at most 50 pM. In a further embodiment, the EC50 is at most 40 pM, such as at most 30 pM, such as at most 20 pM, such as at most 10 pM. The potency of a compound with an EC50 of about 10 pM is equivalent to that of a semaglutide molecule.

As described elsewhere herein, the GLP-1 potency must be balanced with the potency of the EGF (A) analog and therefore in some embodiments, the following may be preferred: GLP-1 analogues that are potent GLP-1 such that the potency of the GLP-1 analogue or a compound comprising the GLP-1 analogue is at least 2 times lower than that of a Soma Valley such as a peptide system, such as at least 5 Times, such as at least 10 times, such as at least 25 times, such as at least 50 times, such as at least 100 times (compared to Soma Valley such as peptides). It may even be preferred that its efficacy is reduced compared to wt GLP-1.

In one embodiment, the EC50 of the GLP-1 analogue or a compound comprising the GLP-1 analogue (as measured in C1, without HSA) is at least 25 pM, such as at least 50 pM, such as at least 75 pM, such as At least 100 pM, such as at least 250 pM, or such as at least 500 pM.

In such an embodiment, the EC50 of the GLP-1 analogues or compounds containing the GLP-1 analogues as measured as described in (C1, without HSA) is at most 500 pM, such as at most 400 pM, such as at most 300 pM, Such as up to 200pM, such as up to 100pM, such as up to 50pM

In a further embodiment, the EC50 of the GLP-1 analogue or a compound comprising the GLP-1 analogue (as measured in C1 without HSA) is 20-1000 pM, such as 50-500 pM, such as 100 -250pM, such as 75-100pM.

In a further embodiment, the EC50 of the GLP-1 analog or a compound comprising the GLP-1 analog (as measured in C1 without HSA) is 20-800 pM, such as 20-600 pM, such as 20 -400pM, such as 20-200pM or such as 20-100pM

Alternatively, the EC50 of the GLP-1 analogue or a compound comprising the GLP-1 analogue (as measured in C1 without HSA) is 200-1000pM, such as 300-800pM or 400-600pM or 250-750pM or 300-500pM.

When evaluating effectiveness in the presence of HSA, the above effectiveness considerations are also relevant.

In one embodiment, the EC50 of the GLP-1 analog or a compound comprising the GLP-1 analog (as measured as described in C1 at 1% HSA) is at least 500 pM, such as at least 750 pM, such as at least 1000 pM, such as at least 1500 pM, or such as at least 2000 pM.

In such an embodiment, the EC50 of the GLP-1 analogues or compounds containing the GLP-1 analogues, as described in (C1, under 1% HSA) is at most 2500 pM, such as at most 2000 pM, such as at most 1500pM, such as up to 1250pM, or such as up to 1000pM

In a further embodiment, the EC50 of the GLP-1 analogue or a compound comprising the GLP-1 analogue (as measured as described in C1, at 1% HSA) is 500-2500pM, such as 500-2000pM , Such as 500-1500pM, such as 500-1000pM.

In a further embodiment, the EC50 (as measured as described in C1, at 1% HSA) of the GLP-1 analog or a compound comprising the GLP-1 analog is 750-2500pM, such as 1000-2500pM , Such as 1500-2500pM, such as 2000-2500pM or such as 1800-2500pM

Alternatively, the EC50 of the GLP-1 analogue or a compound containing the GLP-1 analogue (as measured in C1, at 1% HSA) is 500-2500pM, such as 750-2000pM or such as 1000 -2000pM or 1500-2000pM. This GLP-1 efficacy can be reduced to allow full binding to PCSK9 while reducing GLP-1 related side effects such as (but not limited to) nausea.

In-tube binding affinity of GLP-1 analogues and compounds containing GLP-1 analogues can optionally be tested in the in-tube binding assay described in C2 and has the same function as wt GLP-1 or Soma Valley peptides The affinity of the sexual compound is around 1 nM when tested in the presence of low HSA.

In one embodiment, the GLP-1 analog or a compound comprising the GLP-1 analog has an IC50 of at most 200 nM in a test tube binding assay. In a further embodiment, the IC50 is at most 100 nM, such as at most 75 nM, such as at most 50 nM, such as at most 25 nM, such as at most 10 nM, such as at most 5 nM. In some embodiments, the following may be preferred: have a binding that is lower than the binding of the Soma Valley, such as peptides, so that the GLP-1 analogue or the compound containing the GLP-1 analogue has a binding that is greater than that of the Soma Valley For example, peptides are reduced at least 5-fold, such as at least 10-fold, such as at least 25-fold, such as at least 50-fold, such as at least 100-fold (compared to Soma Valley such as peptides). The following may be even better: the binding affinity is reduced compared to the wt GLP-1 line.

In one embodiment, the IC50 (as measured as described in C2, no HSA) of the GLP-1 analogues or compounds containing GLP-1 analogues is at least 1 nM, such as at least 5 nM, such as at least 10 nM, such as at least 25nM, such as at least 50nM, such as at least 100nM.

In such an embodiment, the IC50 of such GLP-1 analogs or compounds containing GLP-1 analogs as described in (C2, no HSA) is at most 200 nM, such as at most 100 nM, such as at most 75 nM, such as at most 50nM, such as at most 25nM, such as at most 15nM, such as at most 10nM, such as at most 5nM.

In a further embodiment, the IC50 (as measured as described in C2, without HSA) of the GLP-1 analogue or a compound comprising a GLP-1 analogue is 0.1-200 nM, such as 1-100 nM, such as 5- 75nM, such as 5-50nM.

The above considerations are relevant when assessed in the absence of HSA. This GLP-1 binding can be reduced to allow full binding to PCSK9 while reducing GLP-1 related side effects such as (but not limited to) nausea.

The GLP-1 efficacy may alternatively or additionally be measured in vivo by measuring the efficacy of GLP-1 analogs and compounds comprising GLP-1 analogs on blood glucose and / or body weight. The decrease in blood glucose and / or body weight can be measured in suitable models such as db / db mice as described in C7 and DIO rats as described in C8.

In one embodiment, a GLP-1 analog or a compound comprising a GLP-1 analog has the ability to reduce blood glucose in a db / db mouse as described in C7 herein. The efficacy can be estimated based on the area under the curve of △ blood glucose from 0 to 24 hours (AUC △ BG _24h ) and an effective dose of 50% (ED50, which gives the response of GLP-1 derivatives in the middle of the baseline and maximum efficacy Dose) is calculated for AUC ΔBG _24h .

In one embodiment, the following is preferred: the GLP-1 analog or the compound containing the GLP-1 analog has an EC50 AUC ΔBG of less than 15 nmol / kg for _{24 h} . In one embodiment, the EC50 AUC ΔBG _24h is between 1-15, such as 2-12 or such as 5-10 nmol / kg.

The ability to lose weight can be similarly assessed using DIO rats as described in C8.

In one embodiment, when administered at 300 nmol / kg / day and measured after 21 days, the GLP-1 analog or a compound comprising a GLP-1 analog can reduce body weight to at least 95% of the baseline BW.

In one embodiment, when administered at 300 nmol / kg / day and measured after 21 days, the GLP-1 analog or a compound comprising a GLP-1 analog is capable of reducing body weight to at least 90% of the baseline BW.

EGF (A) function- (PCSK9i)

As described herein, the EGF (A) analog is a variant of the LDL-R (293-332) EGF (A) peptide defined by SEQ ID NO: 1. The EGF (A) analog is defined herein as a peptide comprising an amino acid sequence of the analog of SEQ ID NO: 1.

Thus the EGF (A) analogue preferably has the ability to bind to PCSK9. In particular embodiments, the EGF (A) analogs have improved ability to bind to PCSK9, such as compared to natural LDL-R (293-332) (natural EGF (A)) or compared to other PCSK9 Binding compound.

EGF (A) analogs may further have the ability to inhibit the binding of PCSK9 to LDL-R. In one embodiment, the EGF (A) analog is a PCSK9 inhibitor. In one embodiment, the EGF (A) analog inhibits binding to the PCSK9 human low density lipoprotein receptor (LDL-R).

Such a combination can be used for the evaluation described in section C3 herein, which measures the ability of a test compound to competitively inhibit the binding of PCSK9 to human LDLR. Because of their ability to inhibit the interaction of PCSK9 with LDL-R, such compounds are known as PCSK9 inhibitors.

In one embodiment, the EGF (A) analog and the compound (fusion polypeptide or derivative) comprising the EGF (A) analog of the present invention are PCSK9 inhibitor compounds or PCSK9 inhibitors for short. In one embodiment, the invention is a compound comprising an EGF (A) analog of SEQ ID NO .: 1, wherein the analog is capable of inhibiting the binding of PCSK9 to the human low density lipoprotein receptor (LDL-R).

In one embodiment, the EGF (A) analog and a compound comprising the analog) have improved ability to bind to PCSK9 as compared to EGF (A) LDL-R (293-332) (SEQ ID 1). Because wt sequences have relatively poor inhibitory functions, EGF (A) analogs can also be compared. In one embodiment, the EGF (A) analog (and the compound containing the analog) has an EGF (A) compared to [299A, 301L, 307I, 309R, 310K] EGF (A) defined by SEQ ID NO.:2. Improved ability to incorporate PCSK9. The effect measured in ELISA assay provided in the reported K _i EGF compound (A) analogues (A) comprise EGF analogs or apparent affinity and as low Ki system described in C3 have potent inhibitory Features of functional compounds.

In one embodiment, the K _i (as measured in the PCSK9-LDL-R binding competitive ELISA analysis (Section C3)) of the EGF (A) analogs and compounds containing the analogs is below 50 nM, Such as below 25nM or such as below 10nM. In a further embodiment, the K _i (as measured in the PCSK9-LDL-R binding competitive ELISA analysis (section C3)) of the EGF (A) analog and a compound comprising the analog is below 8.0 nM , Such as below 5.0 nM, such as below 2.5 nM or even below 2.0 nM. In one embodiment, the K _i (as measured in the PCSK9-LDL-R binding competitive ELISA analysis (section C3)) of the EGF (A) analog and a compound comprising the analog is 0.1-10.0 nM, Such as 0.1-8.0nM or 0.1-5.0nM.

The functional characteristics of EGF (A) analogs and compounds containing them can be further defined by their ability to improve LDL uptake (such as described in section C4 herein).

In one embodiment, the EGF (A) analog and a compound comprising the analog increase LDL uptake in the presence of PCSK9. In one embodiment, the EGF (A) analog and a compound comprising the same are capable of reversing or reducing PCSK9-mediated reduction in LDL uptake.

In one embodiment, the EGF (A) analog and a compound comprising the analog have an EC50 of less than 1500 nM (such as less than 1000 nM or such as less than 500 nM) (as measured in an LDL uptake analysis).

The efficacy of EGF (A) analogs and compounds containing analogs on blood cholesterol can be assessed in suitable models, such as by studies in DIO rats as described in section C8 herein. The study included several administrations of the test compound and the efficacy was therefore dependent on the dose and frequency of administration. In this study, the efficacy of low, high and very high doses were evaluated after 21 days.

In one embodiment, the EGF (A) analog or a compound comprising the EGF (A) analog is capable of reducing cholesterol by at least 0.5 mmol / L when administered at 30 nmol / kg / day and measured after 21 days. In a further embodiment, when administered at 30 nmol / kg / day and measured after 21 days, cholesterol levels are reduced by at least 0.6 or such as 0.8 mmol / L.

In one embodiment, the EGF (A) analog or a compound comprising the EGF (A) analog is capable of reducing cholesterol by at least 0.8 mmol / L when administered at 300 nmol / kg / day and measured after 21 days. In a further embodiment, when administered at 300 nmol / kg / day and measured after 21 days, cholesterol levels are reduced by at least 1.0 or such as 1.2 mmol / L.

Bifunctional

As described herein above, different functionalities are associated with the two analogs (the GLP-1 analog and the EGF (A) analog). When the two are combined, in the compound of the present invention, the functionality of each analog is preferably maintained, that is, the GLP-1 analog has the ability to stimulate the GLP-1 receptor and the EGF (A) is similar Compounds that competitively bind PCSK9 and further include both of these analogs have both of these functionalities. The functionality of such compounds can be tested in the assays described herein to test GLP-1 and EGF (A) functionality.

In one embodiment, the compounds are referred to as bifunctional molecules.

In order to obtain compounds suitable for therapeutic use, these functionalities must be balanced to obtain the desired level of activity of both PCSK9 inhibitors and GLP-1 receptor agonists. In one embodiment, the compound is a GLP-1 receptor agonist as described herein above. In one embodiment, the compound is a PCSK9 inhibitor as described herein above. The measurement of GLP-1 receptor potency is described in Section C1 and the binding affinity for GLP-1 analogs is described in Section C2, and these functionalities need to be similar for the inclusion of GLP-1 analogs and EGF (A) The compounds are equally relevant.

Similarly, the functionality of EGF (A) analogs has been described in the section on EGF (A) function and in the analyses described in sections C3, C4, and C6 of this document.

The compounds according to the invention (comprising GLP-1 analogs and EGF (A) analogs) are monovalent for each of these analogs, i.e. the compounds include an EGF (A) analog and a GLP- 1 analog. To balance GLP-1 receptor agonist function and PCSK9 inhibitor function, these analogs can be individually selected to obtain suitable levels of both activities. High doses of GLP-1 receptor agonists that may cause side effects (such as nausea) are well known and reduce GLP-1 potency while ensuring good PCSK9 inhibitory function to obtain a proper balance of both activities at the same plasma concentration. of.

In one embodiment, the GLP-1 potency is reduced compared to GLP-1 (3-37) or Soma Valley as a peptide system, as described herein above. In such an embodiment, the EC50 measured by in-tube cre luc analysis (chapter C1, without HSA) is at least 10 pM

In one embodiment, the apparent K _i measured by a competitive ELISA (Section C3) is below 50 nM,)

In one embodiment, the ratio of the apparent EGF (A) Ki (C3) to the GLP-1 efficacy (C1, no HSA) is at most 5000, such as at most 4000, such at most 3000, such at most 2000, or at most 1000 .

In one embodiment, the ratio of the apparent EGF (A) Ki (C3) to the GLP-1 efficacy (C1, no HSA) is at most 1000, such as at most 800, such at most 600, such at most 400, or at most 200 .

In one embodiment, the ratio of the apparent EGF (A) Ki (C3) to the GLP-1 efficacy (C1, no HSA) is at most 200, such as at most 150, such as at most 100, such as at most 50.

In order to confirm that the compound is indeed bifunctional, it is better to assess the functionality, as described herein, which can be done in DIO rats as described in section C8 of this document, where the effects on both body weight and cholesterol can be measured efficacy.

In one embodiment, the compound is capable of reducing cholesterol and weight at least equal to GLP-1 / EGF (A) compound # 41 in an in vivo rat study as described in section C8 herein.

In one embodiment, the compound is capable of reducing cholesterol by at least 0.5 mmol / L when administered at 30 nmol / kg / day and measured after 21 days.

In a further embodiment, the cholesterol level is reduced by at least 0.6 or such as 0.8 mmol / L when administered at 30 nmol / kg / day and measured after 21 days.

In one embodiment, the compound is capable of reducing cholesterol by at least 0.8 mmol / L when administered at 300 nmol / kg / day and measured after 21 days.

In a further embodiment, the cholesterol level is reduced by at least 1.0 or such as 1.2 mmol / L when administered at 300 nmol / kg / day and measured after 21 days.

In one embodiment, the compound is capable of reducing body weight to at least 95% of baseline BW when administered at 300 nmol / kg / day and measured after 21 days.

In one embodiment, the compound is capable of reducing body weight to at least 90% of baseline BW when administered at 300 nmol / kg / day and measured after 21 days.

derivative

The term "derivative" is used herein in the context of a bifunctional compound to mean a chemically modified bifunctional compound in which one or more substituents have been covalently attached to the compound.

As described herein above, this substituent is covalently attached to the compounds. Various ways of attaching a substituent to a polypeptide are known, such as by attaching a substituent through an N-terminal, C-terminal, or internal amino acid residue.

In one embodiment, the compound may include one or more substituents. In one embodiment, the compound contains one or two substituents. In one embodiment, the compound has one or two substituents. In one embodiment, the compound has a substituent. In one embodiment, the compound has two substituents.

In an embodiment in which the compound has two substituents, it is preferred that the two substituents are identical.

In one embodiment, the one or two substituents are attached to a nitrogen atom of the peptide backbone. In one embodiment, the one or two substituents are attached to an amine group of the peptide backbone. In one embodiment, the one or two substituents are attached to the epsilon nitrogen of one or two Lys residues.

In one embodiment, the two substituents are attached to different Lys residues of the peptide backbone. In one embodiment, the two substituents are attached to the ε-nitrogens of different Lys residues in the peptide backbone.

As described herein above, a fusion polypeptide or peptide backbone comprising a GLP-1 analog, a peptide spacer, and an EGF (A) analog or a derivative thereof may have one or more Lys Residues. Various examples of GLP-1 analogs, peptide spacers, and EGF (A) analogs with different numbers of Lys residues have been described herein above, and such sequences can be combined to have a sequence with exactly one or two Lys residue fusion peptide or peptide backbone.

In one embodiment, the peptide backbone has one or two Lys residues. In one embodiment, the peptide backbone comprises only one Lys residue. In one embodiment, the peptide backbone comprises exactly two Lys residues.

In one embodiment, the peptide backbone comprises a GLP-1 analogue comprising one or two Lys residues. In one embodiment, the peptide backbone comprises a GLP-1 analogue comprising only one Lys residue. In one embodiment, the peptide backbone comprises a GLP-1 analogue comprising exactly two residues.

In one embodiment, the peptide backbone comprises an EGF (A) analogue comprising one or two Lys residues. In one embodiment, the peptide backbone comprises an EGF (A) analog containing only one Lys residue. In one embodiment, the peptide backbone comprises an EGF (A) analogue comprising exactly two Lys residues.

In one embodiment, the peptide backbone comprises a peptide spacer comprising one or two Lys residues. In one embodiment, the peptide backbone comprises a peptide spacer comprising only one Lys residue. In one embodiment, the peptide backbone comprises a peptide spacer comprising exactly two residues.

In one embodiment, the purpose of the substituent is to improve the functionality of the peptide.

In one embodiment, the substituent increases the half-life of the compound such that the plasma half-life of the derivative comprising the peptide backbone and the substituent has an increased half-life compared to the half-life of the peptide backbone, as described herein (section C5, Table 5).

Methods for determining the half-life in different species are well known in the art and are exemplified herein for mini-pigs (section C5).

In one embodiment, the derivative according to the invention has a half-life of more than 12 hours.

In one embodiment, the derivative according to the invention has a half-life of more than 24 hours (such as 36 hours or more or 48 hours or more) when measured in a minipig after subcutaneous or intravenous administration.

Substituent

The term "substituent" refers to a moiety attached to a polypeptide through an amino acid residue by substitution of an atom normally present at the same position. Often, replacement of the hydrogen atom of the substituent group, such as an amine group (-NH ₂₎ of hydrogen. This substituent is therefore covalently attached to the peptide or polypeptide portion. According to the present invention, it is preferred that the moiety (for example, the substituent) has no effect on the functionality of the peptide or has a minimal effect while adding other beneficial properties, such as increasing stability or increasing half-life.

In one embodiment, the half-life extending substituent is a protein moiety. In further such embodiments, the protein portion may include human albumin, an Fc-domain, or an unstructured protein elongation. In a further embodiment, the protein portion can be fused to one of the analogs. In a further embodiment, the protein portion is an Fc domain and the Fc domain is fused to the GLP-1 analog or the EGF (A) analog. When preparing an Fc fusion, the resulting compound will usually be bivalent, as the two Fc-polypeptides will form an Fc-domain.

In one embodiment, the substituent is not a protein moiety.

In one embodiment, the substituent is not fused to the protein portion of the peptide backbone.

In another embodiment, the substituent is a non-proteinaceous moiety.

In a specific embodiment, the substituent can form a non-covalent complex with albumin, thereby promoting circulation of the derivative in the bloodstream, and also has the effect of extending the action time of the derivative. In a particular embodiment, the substituent is capable of prolonging the action time of the derivative without substantially reducing its binding ability to the PCSK9 and / or GLP-1 receptor.

In one embodiment, the derivative comprises a half-life extending substituent. Various half-life prolonged substitutions are based on systems that are widely known in the art and specifically include albumin binders (as described further below) containing fatty acid groups, and such albumin binders are non-protein substituents.

The substituent contains at least one fatty acid group.

In a particular embodiment, the fatty acid group comprises a carbon chain containing at least 8 continuous -CH ₂ -groups. In one embodiment, the fatty acid group comprises at least 10 continuous -CH ₂ -groups, such as at least 12 continuous -CH ₂ -groups, at least 14 continuous -CH ₂ -groups, at least 16 Continuous -CH ₂ -groups, at least 18 continuous -CH ₂ -groups.

In one embodiment, the fatty acid group comprises 8-20 consecutive -CH ₂ -groups. In one embodiment, the fatty acid group comprises 10-18 consecutive -CH ₂ -groups. In one embodiment, the fatty acid group comprises 12-18 consecutive -CH ₂ -groups. In one embodiment, the fatty acid group comprises 14-18 consecutive -CH ₂ -groups.

In the case where the derivative contains two substituents, an increased half-life can be obtained with a shorter fatty acid group, so in embodiments where the derivative contains two substituents, the fatty acid groups may contain at least 8 consecutive -CH ₂ -groups, such as at least 10 continuous -CH ₂ -groups, such as at least 12 continuous -CH ₂ -groups, at least 14 continuous -CH ₂ -groups, at least 16 continuous -CH ₂ -groups, at least 18 consecutive -CH ₂ -groups.

In a further embodiment in which the derivative comprises two substituents, the substituents each comprise a fatty acid group comprising 8 to 18 consecutive -CH ₂ -groups. In a further such embodiment, the fatty acid groups include 10-18 continuous -CH ₂ -groups, such as 12-18 continuous -CH ₂ -groups, such as 14-18 continuous -CH ₂ -Group.

The term "fatty acid group" is used herein as a chemical group that may be considered to include at least one functional group that is a Brewster-Loreic acid having a pKa of <7.

In one embodiment, the substituent comprises at least eight consecutive -CH ₂ -groups and at least one functional group (FG) having a pKa of <7. Non-limiting examples of such functional groups that are Brewster-Loreic acid include carboxylic acids (including carboxyphenoxy).

In one embodiment, the fatty acid contains a carbonyl group based on the opposite end of its functional group (its acid), so the fatty acid group may also be referred to as a diacid.

In one embodiment, the term "elongator" can be used to describe the fatty acid group of the terminal portion of the substituent responsible for extending the half-life of the compound.

In one embodiment, the extender may be defined by: Chem. 1: HOOC- (CH ₂ ) _n -CO- *, where n is an integer ranging from 8-20, which can also be considered as C (n +2) Diacid is defined as the following: Chem.1b: Where n is an integer ranging from 8-20.

In one embodiment, the extender may be defined by: Chem. 2: HOOC- (C ₆ H ₄ ) -O- (CH ₂ ) _m -CO- *, where m is an integer ranging from 8-11 or It is defined as the following: Chem.2b: Where the carboxyl group is at position 2, 3 or 4 of the (C ₆ H ₄ ) group in Chem. 3 and where m is an integer ranging from 8-11.

In one embodiment, the extender may be defined by Chem1, Chem 1b, Chem 2 or Chem 2b as defined above.

In one embodiment, a substituent according to the invention comprises one or more linker elements. These linker elements can be connected to each other and can be connected to the extender via a amide bond and are referred to as "Z" (see further below).

As further defined herein below, the number of linker elements can be up to six, which is referred to as -Z1-Z2-Z3-Z4-Z5-Z6-, where Z1 is associated with the extender (Pro-) The linked and final Z element is linked to the peptide. In this example, the substituent may be referred to as Pro-Z1-Z2-Z3-Z4-Z5-Z6-. The above symbol * thus indicates the attachment point to Z1, which is nitrogen when bonded through a amide bond. In the embodiment in which the Z1 bond (see below), the symbol * indicates the attachment point of nitrogen to the Z element next door.

In one embodiment, the substituent is defined by: Pro-Z1-Z2-Z3-Z4-Z4-Z5-Z6-, wherein Pro- is selected from Chem1, Chem 1b, Chem 2 and Chem 2b and wherein n It is an integer ranging from 8-20 and m is an integer ranging from 8-11.

In a particular embodiment, n is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 and m is 8, 9, 10 in Chem. 1 or 1b. Or 11.

The term "bond" is used herein to mean a covalent bond. When the linker element of Z1-Z6 is defined as a bond, it is equivalent to the case where the component does not exist. It indicated below may be understood to Z1-Z by any of the lines ₆ do not exist any person Z1-Z6 of a key in the system herein. Logically, "one key" cannot be followed by "one key". Reference to "one-click" in this context therefore means that the previous Z element is covalently connected to the next Z element that is not "one-click" (or does not exist).

The linker elements Z1-Z6 are individually selected from chemical moieties capable of forming amido bonds, including amino acid-like moieties, such as Glu, γGlu (which is also known as γ Glu or gGlu and are made of * -NH-CH- (COOH ) -CH ₂ -CH ₂ -CO- *) Gly, Ser, Ala, Thr, Ado, Aeep and Aeeep and other parts as described below.

In one embodiment, the Z1 element is as needed. In one such embodiment, the Z1 element is selected from Chem. 3: * -NH-CH _2- (C ₆ H ₁₀ ) -CO- * or Chem. 3b: , And one click.

Chem.3 can also be called Trx, which means Tranexamic acid trans-4- (aminomethyl) cyclohexanecarboxylic acid, of which Chem 3. covers ortho- (1,2), meta- (1,3) and pair- (1,4) forms, while Chem 3b. Specifies the pair- (1,4) form.

In one embodiment, Z2 is selected from γGlu, Glu, or a single bond. In one embodiment, Z2 is γGlu.

In one embodiment, the Z3, Z4, Z5, and Z6 lines (independent of each other) are selected from Glu, γGlu, Gly, Ser, Ala, Thr, Ado, Aeep, and Aeeep, and a bond.

Glu, Gly, Ser, Ala, and Thr are amino acid residues which are widely known in the art.

γGlu is defined by Chem. 4: * -NH-CH (COOH)-(CH ₂ ) ₂ -CO- *, which is the same as Chem. 4b: It can also be called γGlu.

Ado is defined by Chem. 5: * -NH- (CH ₂ ) ₂ -O- (CH ₂ ) ₂ -O-CH ₂ -CO- *, which can also be referred to as 8-amino-3,6- Dioxooctanoic acid and it is the same as: Chem. 5b:

Aeep is defined by Chem. 6: * NH-CH ₂ CH ₂ OCH ₂ CH ₂ OCH ₂ CH ₂ CO *, which can also be considered as the following Chem. 6b:

Aeeep is defined by Chem. 7: * NH-CH ₂ CH ₂ OCH ₂ CH ₂ OCH ₂ CH ₂ OCH ₂ CH ₂ CO *, which can also be considered as the following: Chem. 7b:

In one embodiment, the Z ₃ , Z ₄ , Z ₅ and Z ₆ series (independent of each other) are selected from Glu, γGlu, Gly, Ala, Ado, Aeep, and Aeeep, and a bond.

In one embodiment, the Z ₃ , Z ₄ , Z ₅ and Z ₆ series (independent of each other) are selected from Glu, γGlu, Gly, Ala, Ado and a bond.

In one embodiment, the Z ₃ , Z ₄ , Z ₅ and Z ₆ series (independent of each other) are selected from Glu, γGlu, Gly, Ado and a bond.

In one embodiment, the Z ₃ , Z ₄ , Z ₅ and Z ₆ series (independent of each other) are selected from γGlu, Gly, Ado and a bond.

In one embodiment, the Z ₃ , Z ₄ , Z ₅ and Z ₆ series (independent of each other) are selected from γGlu, Ado and a bond.

In one embodiment, the (etc.) substituent is selected from the group of substituents defined by # 1 to # 14 below.

Substituent # 1 is defined by Chem. 6: HOOC- (CH ₂ ) ₁₆ -CO-γGlu-Ado-Ado- *, which is the same as the following: Chem. 6b:

Substituent # 2 is defined by Chem. 7: HOOC- (CH ₂ ) ₁₆ -CO-γGlu-Ado-Ado-Ado-Ado- *, which is the same as the following: Chem. 7b:

Substituent # 3 is defined by Chem. 8: HOOC- (CH ₂ ) ₁₆ -CO-γGlu-Ado *, which is the same as the following: Chem. 8b:

Substituent # 4 is defined by Chem. 9: HOOC- (CH ₂ ) ₁₆ -CO-γGlu- *, which is the same as the following: Chem. 9b:

Substituent # 5 is defined by Chem. 10: HOOC- (CH ₂ ) ₁₈ -CO-γGlu-Ado-Ado- *, which is the same as the following: Chem. 10b:

Substituent # 6 is defined by Chem. 11: HOOC- (CH ₂ ) ₁₈ -CO-Trx-γGlu-Ado-Ado- *, which is specifically described as: Chem. 11b:

Substituent # 7 is defined by Chem. 12: HOOC- (CH ₂ ) ₁₈ -CO-Trx-γGlu-γGlu-γGlu-γGlu- *, which is specifically described as: Chem. 12b:

Substituent # 8 is defined by Chem. 13: HOOC- (CH ₂ ) ₁₈ -CO-Trx-γGlu-Ado-Ado-Ado- *, which is specifically described as: Chem. 13b:

Substituent # 9 is defined by Chem. 14: HOOC- (CH ₂ ) ₁₈ -CO-Trx-γGlu-γGlu- *, which is specifically described as: Chem. 14b:

Substituent # 10 is defined by Chem. 15: HOOC- (CH ₂ ) ₁₈ -CO-Trx-γGlu-Ado- *, which is specifically described as: Chem. 15b:

Substituent # 11 is defined by Chem. 16: HOOC- (CH ₂ ) ₁₈ -CO-Trx-γGlu-Ado-Ado-Ado-Ado- *, which is specifically described as: Chem. 16b:

Substituent # 12 is defined by Chem. 17: HOOC- (CH ₂ ) ₁₈ -CO-Trx-γGlu- *, which is specifically described as: Chem. 17b:

Substituent # 13 is defined by Chem. 18: 4-COOH-PhO-C11-γGlu-Ado-Ado- *, which is specifically described as: Chem. 18b:

Substituent # 14 is defined by Chem. 19: HOOC- (CH ₂ ) ₁₄ -CO-γGlu-Ado-Ado- *, which is specifically described as: Chem. 19b:

Bifunctional compound

A number of fusion compounds are described herein and the challenge as described elsewhere is to ensure that both functionality is maintained and balanced. The disclosed compounds include changes in the order of the EGF (A) analog, the spacer, and the GLP-1 analog and the elements.

In one embodiment, the fusion polypeptide comprises the GLP-1 analog at the N-terminus and the EGF (A) analog at the C-terminus, which was found to be important for maintaining GLP-1 functionality.

In a further embodiment, the sequences of the EGF (A) analogs should include at least a 301L mutation, and preferably include one or more of 309R and 309I, as described in detail herein and by SEQ ID NO. : The sequences of the 107 and 108 identified EGF (A) analogs are exemplified (which may also include a 312E mutation to remove wild-type lysine).

In one embodiment, the GLP-1 analog comprises mutations as described herein above, such as residue 8 becomes a mutation such as 8Aib, 8G or 8W, and residue 34 becomes a mutation such as 34R, which allows the substitution The base is attached to K26. Further mutations (such as 30G) may be advantageous for reducing the efficacy of the GLP-1 analog.

In such an embodiment, the compound comprises a GLP-1 analog and an EGF (A) analog, wherein i) the GLP-1 analog is identified by SEQ ID No .: 139, 140, 141, 142, or 164 And ii) the EGF (A) analog is identified by SEQ ID No .: 107, 108109.

In one embodiment, the compound comprises a GLP-1 analog and an EGF (A) analog, wherein i) the GLP-1 analog is identified by SEQ ID No .: 139 or 164, and ii) the EGF (A) The analog is identified by SEQ ID No .: 107 or 108.

In one embodiment, the compound comprises a GLP-1 analog and an EGF (A) analog, wherein i) the GLP-1 analog is identified by SEQ ID No .: 164, and ii) the EGF (A ) Analogs are identified by SEQ ID No .: 108.

In one embodiment, the compound is any of GLP-1 / EGF (A) compounds # 1-74 and 76-314.

In one embodiment, the compound is selected from the group of compounds as defined by GLP-1 / EGF (A) compound # 1-74, 76-314.

In one embodiment, the compound is GLP-1 / EGF (A) compound # 69 and / or # 306. In one embodiment, the compound is GLP-1 / EGF (A) compound # 69. In one embodiment, the compound is GLP-1 / EGF (A) compound # 306.

In one embodiment, the compound is GLP-1 / EGF (A) compound # 41 and / or # 48. In one embodiment, the compound is GLP-1 / EGF (A) compound # 41. In one embodiment, the compound is GLP-1 / EGF (A) compound # 48.

Preparation method

The compounds described herein can be prepared using general general knowledge. The backbone or fusion polypeptide may be provided by chemical synthesis (as described in Method Section A1) or by heterologous expression. The expression vector encoding the fusion polypeptide can be prepared by ordinary molecular biology and a suitable host can be selected. It is also possible to combine methods whereby one part of the main chain is prepared synthetically and the other part of the main chain is prepared by recombinant technology. Its substituents may be attached to the peptide backbone during chemical synthesis or in subsequent reactions with the backbone or portions thereof. Independent of the method of preparation, these compounds are defined by their elements, such as a fusion polypeptide (the peptide backbone) and one or more substituents.

One aspect of the present invention pertains to a method for preparing a fusion polypeptide comprising a GLP-1 analog and an EGF (A) analog as described herein.

One aspect of the invention relates to a method for preparing a derivative of a fusion polypeptide comprising a GLP-1 analog and an EGF (A) analog, the derivative further comprising one or more substitutions covalently attached to the fusion polypeptide base.

Pharmaceutical composition

The present invention also relates to a pharmaceutical composition comprising a compound of the present invention (or a pharmaceutically acceptable salt, amidine, or ester thereof) and a pharmaceutically acceptable excipient. Such a composition can be prepared as known in the art.

The term "excipient" refers generally to any ingredient other than the active therapeutic ingredient. The excipient may be an inert substance, an inactive substance, and / or a non-pharmaceutically active substance. The excipient can be used for various purposes, for example, as a carrier, carrier, diluent, lozenge auxiliary, and / or to improve the administration and / or absorption of the active substance. Non-limiting examples of excipients are: solvents, diluents, buffers, preservatives, tonicity adjusters, chelating agents, and stabilizers. The formulation of pharmaceutically active ingredients and various excipients is known in the art, see, for example, Remington: The Science and Practice of Pharmacy (eg, 19th Edition (1995) and any subsequent editions).

The composition of the invention may be in the form of a liquid formulation (ie, an aqueous formulation containing water). Liquid formulations can be solutions or suspensions. Alternatively, it may be a solid formulation, such as a freeze-dried or spray-dried composition.

The composition of the present invention can be used for parenteral administration, for example, administration is intended to be performed by a subcutaneous, intramuscular, intraperitoneal or intravenous injection through a syringe (pen-type syringe if necessary) or by an infusion pump.

The composition of the present invention may further include a second active ingredient (such as a therapeutic agent), which can simplify administration in the case of combination therapy.

Examples of formulations include liquid formulations, ie, aqueous formulations containing water. Liquid formulations can be solutions or suspensions. Aqueous formulations typically contain at least 50% w / w water, or at least 60%, 70%, 80%, or even at least 90% w / w water.

Alternatively, the pharmaceutical composition may be a solid formulation, such as a freeze-dried or spray-dried composition, which may be used by itself, or a physician or patient may add solvents and / or diluents thereto before use.

The pH in the aqueous formulation may be any one between pH 3 and pH 10, such as about 7.0 to about 9.5; or about 3.0 to about 7.0, such as 7.0 to 9.5, or 3.0 to 7.0.

The pharmaceutical composition may include a buffering agent. The buffering agent may be selected, for example, from sodium acetate, sodium carbonate, citrate, glycine, glycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, disodium hydrogen phosphate , Sodium phosphate, and tris (hydroxymethyl) -aminomethane, dihydroxyethylglycine, tris (hydroxymethyl) methylglycine, malic acid, succinate, maleic acid, fumarate Adipic acid, tartaric acid, aspartic acid, and mixtures thereof.

The pharmaceutical composition may contain a preservative. The preservative can be, for example, selected from phenol, o-cresol, m-cresol, p-cresol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, 2-phenoxyethanol, p- Butyl hydroxybenzoate, 2-phenylethanol, benzyl alcohol, chlorobutanol, and sodium thiomersal, bronopol, benzoic acid, imidurarea, lohexidine, Dehydrated sodium acetate, chlorocresol, ethyl p-hydroxybenzoate, benzonine chloride, chlorphenesine (3-p-chlorophenoxypropane-1,2-diol), and And so on. The preservative can be present at a concentration of 0.1 mg / ml to 20 mg / ml.

The pharmaceutical composition may include an isotonicity agent. The tonicity agents can be, for example, selected from salts (e.g., sodium chloride), sugars or sugar alcohols, amino acids (e.g., glycine, histamine, arginine, lysine, isoleucine, aspartic acid) Amino acid, tryptophan acid, hydroxybutyric acid), alditol (e.g. glycerol (glycerol), 1,2-propanediol (propylene glycol), 1,3-propanediol, 1,3-butanediol) Polyethylene Diols (e.g. PEG400), and mixtures thereof. Any sugar can be used, such as mono-, di-, or polysaccharides, or water-soluble dextran, including, for example, sugar, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, Polytridextrose, dextrin, cyclodextrin, α and β HPCD, soluble starch, hydroxyethyl starch, and carboxymethyl cellulose-Na. Sugar alcohols are defined as C4-C8 hydrocarbons having at least one -OH group and include, for example, mannitol, sorbitol, inositol, galactitol, sweet alcohol, xylitol, and arabitol. In one embodiment, the sugar alcohol additive is mannitol.

The pharmaceutical composition may include a chelating agent. The chelating agent may be, for example, a salt selected from ethylenediaminetetraacetic acid (EDTA), citric acid, and aspartic acid salts, and mixtures thereof.

The pharmaceutical composition may include a stabilizer. The stabilizer may be, for example, one or more oxidation inhibitors, aggregation inhibitors, surfactants, and / or one or more protease inhibitors. The pharmaceutical composition may include a stabilizer selected from a high molecular weight polymer or a low molecular weight compound. The stabilizer may be, for example, selected from polyethylene glycol (e.g. PEG 3350), polyvinyl alcohol (PVA), polyvinyl pyrrolidone, carboxy / hydroxycellulose or derivatives thereof (e.g. HPC, HPC-SL, HPC- L and HPMC), cyclodextrin, sulfur-containing substances such as monothioglycerol, thioacetic acid and 2-methylthioethanol, and various salts (such as sodium chloride).

The pharmaceutical composition may include additional stabilizers, such as (but not limited to) methionine and EDTA, which protect the polypeptide from methionine oxidation, and non-ionic surfactants, which protect the polypeptide from contact with Freeze-thaw or mechanical shear associated aggregation.

The pharmaceutical composition may include one or more surfactants. The term "surfactant" refers to any molecule or ion composed of a water-soluble (hydrophilic) portion and a fat-soluble (lipophilic) portion. The surfactant may be, for example, selected from an anionic surfactant, a cationic surfactant, a nonionic surfactant, and / or a zwitterionic surfactant.

The pharmaceutical composition may include one or more protease inhibitors, such as, for example, EDTA (ethylenediaminetetraacetic acid), and / or benzamidine HCl.

In addition, as needed, the ingredients of the pharmaceutical composition include, for example, wetting agents, emulsifiers, antioxidants, accumulating agents, metal ions, oily carriers, proteins (for example, human serum albumin, gelatin), and / or Zwitterions (for example, amino acids such as betaine, taurine, spermine, glycine, lysine, and histamine).

The derivative or analog may be administered as a pharmaceutical composition. It can be administered to patients in need thereof by various routes known in the art. The route of administration can be, for example, parenteral, epidermal; dermis; transdermal; conjunctiva; ureter; vagina; rectum; and / or eye, tongue; sublingual; cheek; mouth; mouth; stomach; intestine Nose; lungs, such as through the small bronchi, alveoli, or combinations thereof.

The composition can be administered in several dosage forms, such as solutions; suspensions; emulsions; microemulsions; compound emulsions; foams; salves; pastes; plasters; ointments; lozenges; coated lozenges; chewing gum; Emollients; capsules, such as hard or soft gelatin capsules; suppositories; rectal capsules; drops; gels; sprays; powders; aerosols; inhalants; eye drops; ophthalmic ointments; eye lotions Vaginal pessary; vaginal ring; vaginal ointment; injection solution; in situ transformation solution, such as in situ gelation, solidification, precipitation, and in situ crystallization; infusion solution; or as an implant.

The composition may be further mixed in a drug carrier or a drug delivery system, for example, to improve stability, bioavailability, and / or solubility. The composition can also be used in formulations of controlled, sustained, extended, delayed, and / or slow release drug delivery systems.

Medical use

In one aspect, the invention relates to the use of a compound according to the invention for use in the manufacture of a medicament.

The present invention also relates to a compound of the present invention or a pharmaceutical composition thereof for use as a pharmaceutical or in the manufacture of a pharmaceutical.

In one embodiment, the compound of the present invention or a composition thereof can be used in the treatment or prevention of cardiovascular disease and / or cardiovascular risk.

In one embodiment, the compound of the present invention or its composition can be used for i. Improving lipid parameters, such as prevention and / or treatment of dyslipidemia, reducing total serum lipids; reducing LDL-C, increasing HDL; reducing small dense LDL; Lower VLDL; lower triglycerides; lower cholesterol; lower plasma levels of lipoprotein a (Lp (a)); inhibit the production of lipoprotein A (apo (A)); ii. Cardiovascular diseases such as Prevention and / or treatment: Cardiac syndrome X, atherosclerosis, myocardial infarction, coronary heart disease, reperfusion injury, stroke, cerebral ischemia, early heart disease or early cardiovascular disease, left ventricular hypertrophy, coronary artery disease, high Blood pressure, essential hypertension, acute hypertension crisis, cardiomyopathy, cardiac insufficiency, exercise intolerance, acute and / or chronic heart failure, arrhythmia, abnormal heart rhythms, syncope, angina, heart bypass and / or stent resuscitation Occlusion, intermittent claudication (occlusive atherosclerosis), diastolic dysfunction, and / or systolic dysfunction; and / or a decrease in blood pressure, such as a decrease in systolic blood pressure; Treatment of diseases.

The invention also relates to methods for the treatment or prevention of cardiovascular disease and / or cardiovascular risk

The invention further relates to methods for: (i) improving lipid parameters, such as the prevention and / or treatment of dyslipidemia, reducing total serum lipids; increasing HDL-C; reducing LDL-C, reducing small dense LDL-C Lower VLDL-C; lower triglycerides; lower cholesterol; lower plasma levels of lipoprotein a (Lp (a)); inhibit the production of lipoprotein A (apo (A)); (ii) such as Prevention and / or treatment of cardiovascular disease: cardiac syndrome X, atherosclerosis, myocardial infarction, coronary heart disease, reperfusion injury, stroke, cerebral ischemia, early heart disease or early cardiovascular disease, left ventricular hypertrophy, coronary Arterial disease, hypertension, essential hypertension, acute hypertension crisis, cardiomyopathy, cardiac insufficiency, exercise intolerance, acute and / or chronic heart failure, arrhythmia, abnormal heart rhythm, syncope, angina pectoris, heart bypass and Re-occlusion of the stent, intermittent claudication (occlusive atherosclerosis), diastolic dysfunction, and / or systolic dysfunction; and / or a decrease in blood pressure, such as a decrease in systolic blood pressure; cardiovascular disease Treatment; wherein a pharmaceutically active amount of a compound according to the invention is administered.

In some embodiments, the compounds of the present invention are useful in the following medical treatments: i. Prevention and / or treatment of all forms of diabetes, such as: hyperglycemia, type 2 diabetes, glucose intolerance, type 1 Diabetes, non-insulin-dependent diabetes, MODY (Young Adult Diabetes), gestational diabetes, and / or reduction in HbA1C; ii. Delay or prevent the progression of diabetic diseases, such as progression of type 2 diabetes, delayed glucose Intolerance (IGT) progresses to insulin-required type 2 diabetes, delays or prevents insulin resistance, and / or delays the progression of non-insulin-required type 2 diabetes to insulin-required type 2 diabetes; iii. Improves beta cells Functions, such as reducing beta cell apoptosis, increasing beta cell function and / or beta cell mass, and / or for restoring beta cells to glucose sensitivity; iv. Prevention of cognitive disorders and / or neurodegenerative disorders such as And / or treatment: Alzheimer's disease, Parkinson's disease, and / or multiple sclerosis; v. Prevention and / or treatment of abnormal appetite disorders such as obesity, such as by borrowing Obesity induced by reducing food intake, weight loss, appetite suppression, and satiety; treating or preventing binge eating disorder, psychogenic bulimia, and / or the administration of antipsychotics or steroids; stomach exercise Reduction; delayed gastric emptying; increased body movement; and / or prevention and / or treatment of comorbidities such as osteoarthritis and / or obesity in obesity; vi. Prevention and / or treatment of diabetic complications such as Treatment: vascular disease; neuropathy, including peripheral neuropathy; nephropathy; and / or retinopathy; vii. Improve lipid parameters, such as prevention and / or treatment of dyslipidemia, reduce total serum lipids; increase HDL; reduce small dense LDL Lower VLDL; lower triglycerides; lower cholesterol; lower plasma levels of lipoprotein a (Lp (a)) in humans; inhibit the production of lipoprotein A (apo (A)) in vitro and / or in vivo Viii. Prevention and / or treatment of cardiovascular diseases such as: Syndrome X, Atherosclerosis, Myocardial Infarction, Coronary Heart Disease, Reperfusion Injury, Stroke, Cerebral Ischemia, Early Heart Disease Early cardiovascular disease, left ventricular hypertrophy, coronary artery disease, hypertension, essential hypertension, acute hypertension crisis, cardiomyopathy, cardiac insufficiency, exercise intolerance, acute and / or chronic heart failure, arrhythmia, heart Abnormal rhythms, syncope, angina, cardiac bypass and / or stent reocclusion, intermittent claudication (occlusive atherosclerosis), diastolic dysfunction, and / or systolic dysfunction; and / or reduction in blood pressure, such as contraction Lowering of blood pressure; ix. Prevention and / or treatment of gastrointestinal diseases such as: inflammatory bowel disease, short bowel syndrome, or Crohn's disease or colitis; indigestion and / or gastric ulcer; and / or Inflammation, such as psoriasis, psoriatic arthritis, rheumatoid arthritis, and / or systemic lupus erythematosus; x. Prevention and / or treatment of major diseases, such as patients with major diseases, major diseases, multiple kidney disease ( (CIPNP) patients, and / or potential CIPNP patients; prevention of major disease or development of CIPNP; prevention, treatment and / or treatment of systemic inflammatory response syndrome (SIRS) in patients; and / Cure; the patient is prevented or reduced the possibility of bacteremia, sepsis, and / or septic shock during hospitalization; and / or stabilization of blood glucose, insulin balance, and metabolism as needed in patients with intensive care units with acute illness; xi. Prevention and / or treatment of polycystic ovary syndrome (PCOS); xii. Prevention and / or treatment of brain diseases such as cerebral ischemia, cerebral hemorrhage, and / or traumatic brain injury Xiii. Prevention and / or treatment of sleep apnea; and / or xiv. Prevention and / or treatment of abuse such as alcohol and / or substance abuse.

In some embodiments, the indication is selected from the group consisting of (i)-(xiv) (such as indications (i)-(viii), (x)-(xiii), and / or (xiv)) Groups are somehow related to diabetes.

In some embodiments, the indication is selected from (i)-(iii) and (v)-(viii) (such as indications (i), (ii), and / or (iii); or indications ( v), indication (vi), indication (vii), and / or indication (viii)).

In some embodiments, the indication is line (i). In a further particular embodiment, the indication line is (v). In a still further specific embodiment, the indication line is (viii).

In some embodiments, the compounds of the invention can be used in the treatment and / or prevention of all forms of diabetes, including the following: abnormal appetite disorders, cardiovascular disease, gastrointestinal disease, diabetic complications, and / or polycystic Ovarian syndrome; and / or for improving lipid parameters, improving beta cell function, and / or for delaying or preventing the progression of diabetic disease.

The following indications are particularly good: type 2 diabetes and / or obesity.

In some embodiments, the invention relates to a method for weight management. In some embodiments, the present invention relates to a method for reducing appetite. In some embodiments, the invention relates to a method for reducing food intake.

In general, all subjects suffering from obesity are considered to be suffering from overweight. In some embodiments, the invention relates to methods for the treatment or prevention of obesity. In some embodiments, the invention relates to the use of a derivative or analog of the invention for the treatment or prevention of obesity. In some embodiments, the subject suffering from obesity is a human, such as an adult or a pediatric human (including infants, children, and adolescents). Body mass index (BMI) is a measure of body fat based on height and weight. The formula used for the calculation is BMI = weight in kilograms / (height in meters) ² . Human subjects suffering from obesity may have BMI of 30; this subject may also be referred to as obese. In some embodiments, a human subject suffering from obesity may have 35 BMI or range in BMI from 30 to <40. In some embodiments, the obesity is severely obese or morbidly obese, wherein the human subject may have BMI of 40.

In some embodiments, the present invention relates to a method for the treatment or prevention of overweight, if necessary in the presence of at least one weight-related comorbidity.

In some embodiments, the present invention relates to the use of a compound of the present invention for the treatment or prevention of overweight, if necessary in the presence of at least one weight-related comorbidity. In some embodiments, the subject suffering from overweight is a human, such as an adult or a pediatric human (including infants, children, and adolescents). In some embodiments, a human subject suffering from overweight may have 25 BMI, such as BMI of 27. In some embodiments, a human subject suffering from being overweight has a BMI ranging from 25 to <30 or ranging from 27 to <30. In some embodiments, the weight-related comorbidity is selected from the group consisting of hypertension, diabetes (such as type 2 diabetes), dyslipidemia, high cholesterol, and obstructive sleep apnea.

In some embodiments, the invention relates to a method for weight loss. In some embodiments, the invention relates to the use of a compound of the invention for weight loss. Humans who want to experience weight loss according to the present invention may have 25 BMI, such as BMI of 27 or 30 BMI. In some embodiments, a human who is going to experience weight loss according to the present invention may have 35 BMI or BMI of 40. The term "weight loss" may include treatment or prevention of obesity and / or overweight.

In a further embodiment, the present invention relates to the use of a compound according to the present invention for the treatment or prevention of diabetes and cardiovascular disease or cardiovascular risk as mentioned above, which solves two diseases or disorders by one drug.

In one embodiment, the invention relates to a method of treatment as described above, comprising the step of administering a therapeutically effective dose of a compound according to the invention to a patient in need thereof.

The dose to be administered may be determined individually and the dose may be less than 50 mg per week, such as 10-15 mg per week, or 70-100 mg / month, depending on the particular pharmaceutical compound selected and the method of administration.

Although certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those skilled in the art. Therefore, it should be understood that the appended embodiments are intended to cover all such modifications and alterations which fall within the true spirit of the invention.

Implementation

1. A compound comprising a GLP-1 analogue and an EGF (A) analogue, wherein i. The GLP-1 analogue is an analogue of GLP-1 (7-37) identified by SEQ ID NO: 137 and ii. The EGF (A) analog is an analog of the EGF (A) domain (293-332) of LDL-R identified by SEQ ID No: 1.

2. The compound according to embodiment 1, wherein the compound has at least one Lys residue.

3. The compound according to embodiment 1, wherein the compound has at least two Lys residues.

4. The compound of embodiment 1, wherein the compound has exactly one or two Lys residues.

5. The compound according to embodiment 1, wherein the compound has exactly one Lys residue.

6. The compound according to embodiment 1, wherein the compound has exactly two Lys residues.

7. The compound according to embodiment 1, wherein the compound comprises a fusion polypeptide.

8. The compound according to embodiment 7, wherein the fusion polypeptide comprises a GLP-1 analog and an EGF (A) analog.

9. The compound according to embodiment 8, wherein the GLP-1 analog is fused to the EGF (A) analog through the C-terminal amino acid residue of the GLP-1 analog.

10. The compound according to embodiment 8, wherein the fusion polypeptide comprises the GLP-1 analog at the N-terminus and the EGF (A) analog at the C-terminus.

11. The compound according to embodiment 8, wherein the EGF (A) analog is fused to the GLP-1 analog through the C-terminal amino acid residue of the EGF (A) analog.

12. The compound according to embodiment 8, wherein the fusion polypeptide comprises the EGF (A) analog at the N-terminus and the GLP-1 analog at the C-terminus.

13. The compound of any one of embodiments 7-12, wherein the fusion polypeptide comprises a peptide spacer.

14. The compound according to embodiment 13, wherein the peptide spacer is composed of 4-80 amino acid residues.

15. The compound according to embodiment 14, wherein the peptide spacer is composed of 4-20 amino acid residues.

16. The compound of embodiment 14, wherein the peptide spacer comprises a Lys residue.

17. The compound of embodiment 14, wherein the peptide spacer does not contain a Lys residue.

18. The compound of embodiment 14, wherein the peptide spacer is selected from the group of peptides identified by SEQ ID NOs 115-126.

19. The compound of embodiment 14, wherein the peptide spacer is selected from the group of peptides identified by SEQ ID NOs 115-136.

20. The compound according to any one of the preceding embodiments, wherein the GLP-1 analog is a GLP-1 receptor agonist.

21. The compound according to any one of the preceding embodiments, wherein the GLP-1 analogue has 1-50 pM, 10-100 pM, 10-50 pM, EC50 of 50-100pM, 100-250pM or 250-1000pM

22. The compound according to any one of the preceding embodiments, wherein the GLP-1 analog is a full GLP-1 receptor agonist.

23. The compound according to any one of the preceding embodiments, wherein the GLP-1 analogue has a comparable to wt GLP-1 in the GLP-1 in-tube potency analysis (without HSA) described in C1. EC50.

24. The compound of embodiment 23, wherein the GLP-1 analog has an EC50 of at most 50 pM.

25. The compound according to any one of the preceding embodiments, wherein the GLP-1 analogue has a GLP-1 in-tube potency assay (without HSA) described in C1 with EC50.

26. The compound of embodiment 25, wherein the GLP-1 analog has an EC50 of 5-15 pM.

27. The compound of any one of the preceding embodiments, wherein the GLP-1 analogue has an EC50 of up to 2500 pM in the GLP-1 in-vitro potency analysis (under 1% HSA) described in C1.

28. The compound of any one of the preceding embodiments, wherein the GLP-1 analogue has an EC50 of at least 500 pM in the GLP-1 in-vitro potency analysis (under 1% HSA) described in C1.

29. The compound according to any one of the preceding embodiments, wherein the GLP-1 analog has the ability to reduce blood glucose in db / db mice as described in C7.

30. The compound according to any one of the preceding embodiments, wherein the GLP-1 analogue has the ability to reduce blood glucose in db / db mice as described in C7 and wherein its EC50 AUC △ BG _24h line Below 15nmol / kg.

31. The compound according to any one of the preceding embodiments, wherein the GLP-1 analog has the ability to reduce body weight in DIO rats as described in C8.

32. The compound according to any one of the preceding embodiments, wherein the GLP-1 analog has the ability to reduce body weight in DIO rats as described in C8 and wherein the GLP-1 analog kg / day administration and reduced body weight to at least 95% of baseline BW when measured after 21 days.

33. The compound according to any one of the preceding embodiments, wherein the GLP-1 analog has the ability to reduce body weight in DIO rats as described in C8 and wherein the GLP-1 analog is treated as 300 nmol / kg / day administration and reduced body weight to at least 90% of baseline BW when measured after 21 days.

34. The compound of any one of the preceding embodiments, wherein the GLP-1 analogue is at least 80, such as 85, such as 90, such as 95% identical to SEQ ID NO .: 137.

35. The compound according to any one of the preceding embodiments, wherein the GLP-1 analogue comprises up to 6 amino acid substitutions compared to SEQ ID NO.:137.

36. The compound according to any one of the preceding embodiments, wherein the GLP-1 analog comprises an amino acid substitution of 8A.

37. The compound according to any one of the preceding embodiments, wherein the GLP-1 analog comprises an amino acid substitution in which 8A becomes G or W

38. The compound of any one of the preceding embodiments, wherein the GLP-1 analog comprises a non-proteinogenic amino acid residue at position 8.

39. The compound according to any one of the preceding embodiments, wherein the GLP-1 analogue comprises a non-proteinogenic amino acid residue Aib at position 8.

40. The compound according to any one of the preceding embodiments, wherein the GLP-1 analogue comprises an amino acid substitution of 8A to G, W or a non-proteinogenic amino acid residue Aib.

41. The compound according to any one of the preceding embodiments, wherein the GLP-1 analog comprises 8Aib.

42. The compound of any one of the preceding embodiments, wherein the GLP-1 analog comprises zero, one, or two Lys residues.

43. The compound according to any one of the preceding embodiments, wherein the GLP-1 analog comprises one or two Lys residues selected from the group consisting of: 12K, 21K, 23K, 24K, 25K, 26K, 27K, 30K, 31K, 32K, 33K, 34K and 36K.

44. The compound of any one of the preceding embodiments, wherein the GLP-1 analog comprises Lys residues 26K and 34K.

45. The compound of any one of the preceding embodiments, wherein the GLP-1 analog comprises a substitution or deletion of one or both of 26K and 34K.

46. The compound according to any one of the preceding embodiments, wherein the GLP-1 analog does not contain 26K.

47. The compound according to any one of the preceding embodiments, wherein the GLP-1 analog comprises a deletion of 26K.

48. The compound according to any one of the preceding embodiments, wherein the GLP-1 analog comprises a 26K amino acid substitution.

49. The compound of any one of the preceding embodiments, wherein the GLP-1 analog comprises 26R.

50. The compound according to any one of the preceding embodiments, wherein the GLP-1 analog comprises an additional Lys residue.

51. The compound of any one of the preceding embodiments, wherein the GLP-1 analog comprises additional Lys selected from the group of 12K, 21K, 23K, 24K, 25K, 27K, 30K, 31K, 32K, 33K and 36K.

52. The compound of any one of the preceding embodiments, wherein the GLP-1 analog comprises exactly one Lys residue selected from the group consisting of 12K, 21K, 23K, 24K, 25K, 26K, 27K, 30K , 31K, 32K, 33K, 34K, and 36K.

53. The compound of any one of the preceding embodiments, wherein the GLP-1 analog comprises exactly two Lys residues selected from the following pairings:

k) 21K and 26K

l) 23K and 26K

m) 24K and 26K

n) 25K and 26K

o) 27K and 26K

p) 30K and 26K

q) 31K and 26K

r) 32K and 26K

s) 33K and 26K

t) 34K and 26K

54. The compound of any one of the preceding embodiments, wherein the GLP-1 analog does not include 34K.

55. The compound of any one of the preceding embodiments, wherein the GLP-1 analog comprises a deletion of 34K.

56. The compound according to any one of the preceding embodiments, wherein the GLP-1 analog comprises a 34K amino acid substitution.

57. The compound of any one of the preceding embodiments, wherein the GLP-1 analog comprises 34R or 34Q.

58. The compound of any one of the preceding embodiments, wherein the GLP-1 analog comprises a deletion of amino acid residues 35-37, 34-37, or 33-37.

59. The compound of any one of the preceding embodiments, wherein the GLP-1 analog comprises 33L.

60. The compound of any one of the preceding embodiments, wherein the GLP-1 analog comprises at least 26 (such as at least 27 or at least 28) amino acid residues.

61. The compound according to any one of the preceding embodiments, wherein the GLP-1 analog comprises one of the amino acid residues 21, 23, 24, 25, 27, 29, 30, 31, 32, and 33 Substituted by amino acids.

62. The compound according to any one of the preceding embodiments, wherein the GLP-1 analog has a sequence as defined by: HX ₈ -EGTX ₁₂ -TSDVSSYLX ₂₁ -GX ₂₃ -X ₂₄ -X ₂₅ -X ₂₆ -X ₂₇ -FX ₂₉ -X ₃₀ -X ₃₁ -X ₃₂ -X ₃₃ -X ₃₄ -X ₃₅ -X ₃₆ -X ₃₇ (SEQ ID NO 187), where X ₈ is A, G, W or Aib, X ₁₂ is F or X ₂₁ is E, G or K, X ₂₃ is Q, G or K, X ₂₄ is A, G, V or K, X ₂₅ is A, G, V or K, X ₂₆ is K Or R, X ₂₇ is E, G or K, X ₂₉ is I, A or V, X ₃₀ is A, G or K, X ₃₁ is W, G or K, X ₃₂ is L, G, T, V, I or K, X ₃₃ is V, G, I, L, K or absent, X ₃₄ is K, R, Q or absent, X ₃₅ is G or absent, X ₃₆ is R, K or absent, And X ₃₇ is G or does not exist.

63. The compound according to any one of the preceding embodiments, wherein the GLP-1 analog is selected from the group of GLP-1 analogs identified by: SEQ ID NO .: 138 to 186, such as SEQ ID NO .: 139-146, 155-162, 164-173, such as SEQ ID NO .: 139-142, 155-162, 164-173, such as SEQ ID NO .: 139, 142, 155-162, 164 -173, such as SEQ ID NO .: 139, 155-162, 164-173, such as SEQ ID NO .: 155-162, 164-173, or such as SEQ ID NO .: 139, and 164.

64. The compound according to any one of the preceding embodiments, wherein the EGF (A) analog is a PCSK9 inhibitor.

65. The compound according to any one of the preceding embodiments; wherein the EGF (A) analog is compared to the EGF (A) domain (293-332) of LDL-R identified by SEQ ID NO.:1 ) Has increased binding affinity to human PCSK9.

66. The compound of any one of the preceding embodiments, wherein the EGF (A) analog is less than 50 nM when measured in a PCSK9-LDL-R binding competitive ELISA assay as described in section C3 Ki (such as below 25nM or such as below 10nM) combines with PCSK9.

67. The compound of any one of the preceding embodiments, wherein the EGF (A) analog is less than 5 nM when measured in a PCSK9-LDL-R binding competitive ELISA assay as described in section C3 Ki combined with PCSK9.

68. The compound according to any one of the preceding embodiments, wherein the EGF (A) analog increases LDL uptake.

69. The compound according to any one of the preceding embodiments, wherein the EGF (A) analog increases LDL uptake in the presence of human PCSK9.

70. The compound of any one of the preceding embodiments, wherein the EGF (A) analog has an EC50 of less than 1000 nM when measured in the LDL uptake analysis described in Section C4.

71. The compound of any one of the preceding embodiments, wherein the EGF (A) analog has an EC50 of less than 500 nM when measured in the LDL uptake analysis described in Section C4.

72. The compound according to any one of the preceding embodiments, wherein the EGF (A) analog reduces blood cholesterol.

73. The compound according to any one of the preceding embodiments, wherein the EGF (A) analog reduces blood cholesterol in DIO rats when evaluated as described in section C8

74. The compound according to any one of the preceding embodiments, wherein the EGF (A) analog reduces blood cholesterol by at least 0.5 mmol / L when administered at 30 nmol / kg / day and measured after 21 days.

75. The compound according to any one of the preceding embodiments, wherein the EGF (A) analog reduces blood cholesterol by at least 0.8 mmol / L when administered at 300 nmol / kg / day and measured after 21 days.

76. The compound of any one of the preceding embodiments, wherein the EGF (A) analogue is at least 80, 85, 90, or such as 95% identical to SEQ ID NO .: 1 line.

77. The compound according to any one of the preceding embodiments, wherein the EGF (A) analog comprises 1-15 amino acid substitutions compared to SEQ ID NO.:1.

78. The compound according to any one of the preceding embodiments, wherein the EGF (A) analog comprises 301L.

79. The compound according to any one of the preceding embodiments, wherein the EGF (A) analog comprises 301L and 309R.

80. The compound according to any one of the preceding embodiments, wherein the EGF (A) analog comprises (wild-type) amino acid residues 295N (Asn), 296E (Glu), 298L (Leu), 302G (Gly) and 310D (Asp).

81. The compound of any one of the preceding embodiments, wherein the EGF (A) analog does not contain any K residues.

82. The compound of any one of the preceding embodiments, wherein the EGF (A) analog does not include 312K.

83. The compound according to any one of the preceding embodiments, wherein the EGF (A) analog comprises 312E, 312D, 312Q, or 312R.

84. The compound of any one of the preceding embodiments, wherein the EGF (A) analogue comprises an amino acid substitution of 301L, 309R, and 312K, such as 312E.

85. The compound of any one of the preceding embodiments, wherein the EGF (A) analog comprises 301L, 310D, and 312K amino acid substitutions, such as 312E.

86. The compound according to any one of the preceding embodiments, wherein the EGF (A) analog comprises 301L and 310D and the peptide does not have a substitution of 299D to G, V or H.

87. The compound according to any one of the preceding embodiments, wherein the EGF (A) analog comprises 321D or 321E.

88. The compound according to any one of the preceding embodiments, wherein the EGF (A) analog comprises 301L, 309R, 312E, and 321E.

89. The compound according to any one of the preceding embodiments, wherein the EGF (A) analog sequence is defined by any of the following: SEQ ID NO .: 19, 21, 73, 107, 108, 109, 110, 111, 112, 113, and 114, such as 107, 108, 109, 110, and 111, such as 107 and 108.

90. The compound according to any one of the previous embodiments 7-89, wherein the fusion polypeptide comprises a GLP-1 analog, a spacer peptide, and an EGF (A) analog.

91. The compound of embodiment 90, wherein the GLP-1 analog is as defined in any of embodiments 20-63.

92. The compound of embodiment 90 or embodiment 91, wherein the EGF (A) analog is as defined in any one of embodiments 64-89.

93. The compound according to embodiment 90, 91 or 92, wherein the spacer peptide is as defined in any one of embodiments 14-19.

94. The compound according to embodiment 90, wherein the fusion polypeptide is selected from the group consisting of a sequence recognized by SEQ ID NO .: 188-384.

95. The compound of any one of embodiments 90-94, wherein the fusion polypeptide comprises up to two lysine residues.

96. The compound according to any one of the preceding embodiments, wherein the compound comprises up to two substituents.

97. The compound according to any one of the preceding embodiments, wherein the compound comprises up to two half-life extending substituents.

98. The compound according to any one of the preceding embodiments, wherein the compound is a derivative comprising a peptide backbone and up to two substituents attached to the backbone.

99. The compound of embodiment 98, wherein the peptide backbone is a fusion peptide as defined in any of embodiments 7-94.

100. The compound according to any one of the foregoing embodiments 96-99, wherein at least one substituent is attached to the GLP-1 analog, the EGF (A) analog, and / or the spacer.

101. The compound of embodiment 100, wherein at least one substituent is attached to the GLP-1 analog.

102. The compound of embodiment 100, wherein at least one substituent is attached to the EGF (A) analog.

103. The compound of embodiment 100, wherein at least one substituent is attached to the spacer.

104. The compound of embodiment 100, wherein at least one substituent is attached via a Lys / K amino acid residue.

105. The compound of embodiment 100, wherein at least one substituent is attached to the GLP-1 analogue via a Lys / K amino acid residue.

106. The compound of embodiment 100, wherein at least one substituent is attached to the GLP-1 analog by a Lys / K amino acid residue selected from the group consisting of: 12K, 21K, 24K, 25K, 26K, 27K, 31K, 32K and 36K.

107. The compound of embodiment 100, wherein at least one substituent is attached to the GLP-1 analogue via 26K.

108. The compound of embodiment 100, wherein at least one substituent is attached to the EGF (A) analog.

109. The compound according to embodiment 100, wherein at least one substituent is attached to the EGF (A) analog by: 292Lys, 293Lys, 294Lys, 299Lys, 300Lys, 303Lys, 305Lys, 306Lys, 309Lys, 311Lys , 312Lys, 313Lys, 314Lys, 315Lys, 316Lys, 318Lys, 320Lys, 321Lys, 322Lys, 323Lys, 324Lys, 325Lys, 326Lys, 327Lys, 328Lys, 329Lys, 330Lys, 332Lys, or 333Lys.

110. The compound according to embodiment 100, wherein at least one substituent is attached to the EGF (A) analog by: 292Lys, 293Lys, 294Lys, 300Lys, 303Lys, 305Lys, 306Lys, 309Lys, 311Lys, 312Lys 313Lys, 314Lys, 316Lys, 318Lys, 321Lys, 322Lys, 323Lys, 324Lys, 325Lys, 326Lys, 327Lys, 328Lys, 329Lys, 330Lys, 332Lys or 333Lys.

111. The compound of embodiment 100, wherein at least one substituent is attached to the EGF (A) analog by: 292Lys, 293Lys, 294Lys, 300Lys, 303Lys, 305Lys, 306Lys, 311Lys, 312Lys, 313Lys , 314Lys, 316Lys, 318Lys, 321Lys, 322Lys, 323Lys, 324Lys, 325Lys, 326Lys, 327Lys, 328Lys, 329Lys, 330Lys, 332Lys or 333Lys.

112. The compound according to embodiment 100, wherein at least one substituent is attached to the EGF (A) analog by: 292Lys, 293Lys, 294Lys, 300Lys, 303Lys, 305Lys, 306Lys, 311Lys, 313Lys, 314Lys , 316Lys, 318Lys, 321Lys, 322Lys, 323Lys, 324Lys, 325Lys, 326Lys, 327Lys, 328Lys, 329Lys, 330Lys, 332Lys, or 333Lys.

113. The compound of embodiment 100, wherein at least one substituent is attached to the EGF (A) analog by: 313Lys, 321Lys, 324Lys, 328Lys, or 333Lys.

114. The compound of embodiment 100, wherein at least one substituent is attached to the peptide spacer.

115. The compound of embodiment 100, wherein at least one substituent is attached to the peptide spacer via a Lys residue.

116. The compound according to embodiment 100, wherein at least one substituent is attached to the peptide spacer through a Lys residue, wherein the spacer is at positions 1, 2, 3, 4, 5, 6, 7 Or 8 a variant of SEQ ID 116 with Lys.

117. The compound of any one of embodiments 96-116, wherein the compound comprises exactly two substituents attached to the fusion peptide.

118. The compound of embodiment 117, wherein one substituent is attached via a GLP-1 analogue as defined in embodiments 105-107 and one substituent is attached to as described in embodiments 115-116 Spacer as defined in either of the above.

119. The compound of embodiment 117, wherein one substituent is attached via an EGF (A) analog as defined in any one of embodiments 109-113 and one substituent is attached to as The peptide spacer as defined in any one of the embodiments 115-116.

120. The compound of embodiment 117, wherein one substituent is attached via a GLP-1 analogue as defined in any one of embodiments 105-107 and one substituent is attached to An EGF (A) analogue as defined in any of modes 109-113.

121. The compound of embodiment 117, wherein the two substituents are attached via a GLP-1 analogue as defined in any one of embodiments 105-108.

122. The compound of embodiment 117, wherein the two substituents are attached via an EGF (A) analogue as defined in any one of embodiments 109-113.

123. The compound according to any one of the preceding embodiments 96-122, wherein the (or other) substituent comprises a fatty acid group (AB).

124. The compound of embodiment 123, wherein the (or other) substituent comprises a fatty acid group selected from the group consisting of: Chem 1 -C (= O)-(CH ₂ ) _n -COOH, wherein n is an integer ranging from 8-20 and Chem 2 -HOOC- (C ₆ H ₄ ) -O- (CH ₂ ) _m -CO- *, where m is an integer ranging from 8-11.

125. The compound of embodiment 123, wherein the (s) selected from the substituent group comprising a diacid as -C (= O) - (CH 2) n -COOH group of a fatty acid, wherein n lines 14-20.

126. The compound of embodiment 123, wherein the (s) selected from substituent group comprises a diacid _{(-HOOC- (C 6 H 4)} -O- (CH 2) m -CO- *) fatty acid group, Where m is an integer ranging from 8-11.

127. The compound of any one of embodiments 123-126, wherein the at least one substituent further comprises at least one linker element.

128. The compound according to any one of embodiments 123-126, wherein the at least one substituent further comprises up to 6 linker elements, which are represented by -Z ₁ -Z ₂ -Z ₃ -Z ₄ -Z ₅ -Z ₆ -call it.

129. The compound according to any one of embodiments 123-126, wherein the at least one substituent further comprises up to 6 linker elements, which are represented by -Z ₁ -Z ₂ -Z ₃ -Z ₄ -Z ₅ -Z ₆ -is called, wherein Z ₁ is connected to the fatty acid group and the last Z element is connected to the peptide backbone.

130. The compound according to any one of embodiments 128 and 129, wherein -Z ₁ is * -NH-CH _2- (C ₆ H ₁₀ ) -CO- * or a single bond.

131. The compound according to any one of embodiments 128 and 130, wherein -Z ₂ -is γGlu, Glu, or a single bond.

132. The compound according to any one of embodiments 128 and 130, wherein -Z ₂ -is γGlu.

133. The compound according to any one of embodiments 128 and 132, wherein the Z ₃ , Z ₄ , Z ₅ and Z ₆ series (independent of each other) are selected from Glu, γGlu, and Ado and a bond.

134. The compound according to any one of embodiments 128 and 132, wherein the Z ₃ , Z ₄ , Z ₅ and Z ₆ series (independent of each other) are selected from γGlu, Ado and a bond.

135. The compound of any one of embodiments 123-134, wherein the at least one substituent comprises a linker comprising -γGlu-Ado-Ado-.

136. The compound of any one of embodiment 123, wherein the at least one substituent is selected from substituents # 1-13, such as selected from substituents # 1-4, # 5-12, # 6- 12 or a group of substituents consisting of substituents # 1, # 5, and # 6.

137. The compound according to any one of the preceding embodiments, wherein the compound is bifunctional.

138. The compound according to any one of the preceding embodiments, wherein the compound is a GLP-1 receptor agonist.

139. The compound according to embodiment 137 or embodiment 138, wherein the compound is 1-50 pM, 10-100 pM, 50-100 pM, 100 in GLP-1 in vitro potency analysis (without HSA) described in C1 EC50 of -250pM or 250-1000pM.

140. The compound according to any one of the preceding embodiments 137-139, wherein the compound is a full GLP-1 receptor agonist.

141. The compound of any one of the preceding embodiments 137-139, wherein the compound has an EC50 comparable to wt GLP-1 in the GLP-1 in-tube potency analysis (without HSA) described in C1 .

142. The compound according to any one of the preceding embodiments 137-139, wherein the compound has an EC50 of at most 50 pM in the GLP-1 in-vitro potency analysis (without HSA) described in C1.

143. The compound of any one of the preceding embodiments 137-139, wherein the compound has an EC50 comparable to Soma Valley peptides in the GLP-1 in-tube potency analysis (without HSA) described in C1 .

144. The compound according to any one of the preceding embodiments 137-139, wherein the compound has an EC50 of 5-15 pM in the GLP-1 in-vitro potency analysis (without HSA) described in C1.

145. The compound according to any one of the preceding embodiments 137-139, wherein the compound has an EC50 of up to 2500 pM in the GLP-1 in-tube potency analysis (under 1% HSA) described in C1.

146. The compound according to any one of the previous embodiments 137-139, wherein the compound has an EC50 of at least 500 pM in the GLP-1 in vitro potency analysis (under 1% HSA) described in C1.

147. The compound according to any one of the previous embodiments 137-146, wherein the compound has the ability to reduce blood glucose in db / db mice as described in C7.

148. The compound according to any one of the previous embodiments 137-146, wherein the compound has the ability to reduce blood glucose in db / db mice as described in C7 and wherein its EC50 AUC △ BG _24h is low At 15nmol / kg.

149. The compound according to any one of the previous embodiments 137-146, wherein the GLP-1 analog has the ability to reduce body weight in DIO rats as described in C8.

150. The compound according to any one of the previous embodiments 137-146, wherein the compound has the ability to reduce body weight in DIO rats as described in C8 and wherein the GLP-1 analogue is capable of reducing body weight to at least 95% of baseline BW (when dosed at 300 nmol / kg / day and measured after 21 days).

151. The compound of any one of the previous embodiments 137-146, wherein the compound has the ability to reduce body weight in DIO rats as described in C8 and wherein the GLP-1 analogue is capable of reducing body weight to at least 90% of baseline BW (when dosed at 300 nmol / kg / day and measured after 21 days).

152. The compound according to any one of the preceding embodiments, wherein the compound is a PCSK9 inhibitor.

153. The compound according to any one of the foregoing embodiments 137-145, wherein the compound has an EGF (A) domain (293-332) of LDL-R identified by SEQ ID NO .: 1 having Increased binding affinity to human PCSK9.

154. The compound of any one of the previous embodiments 137-145, wherein the compound is less than 50 nM (such as such as when measured in a PCSK9-LDL-R binding competitive ELISA assay as described in section C3) Ki below 25nM or such as below 10nM) combines with PCSK9.

155. The compound of any one of the preceding embodiments 137-145, wherein the compound has a Ki of less than 5 nM when measured in a PCSK9-LDL-R binding competitive ELISA assay as described in section C3 Combined with PCSK9.

156. The compound according to any one of the preceding embodiments 137-145, wherein the compound increases LDL uptake.

157. The compound according to any one of the preceding embodiments 137-145, wherein the compound increases LDL uptake in the presence of human PCSK9.

158. The compound of any one of the preceding embodiments 137-152, wherein the compound has an EC50 of less than 1000 nM when measured in the LDL uptake analysis described in Section C4.

159. The compound of any one of the preceding embodiments 137-145, wherein the compound has an EC50 of less than 500 nM when measured in the LDL uptake analysis described in Section C4.

160. The compound according to any one of the preceding embodiments, wherein the compound reduces blood cholesterol when evaluated in a DIO rat as described in section C8

161. The compound of embodiment 160, wherein the compound reduces blood cholesterol by at least 0.5 mmol / L when administered at 30 nmol / kg / day and measured after 21 days.

162. The compound of embodiment 160, wherein the compound reduces blood cholesterol by at least 0.8 mmol / L when administered at 300 nmol / kg / day and measured after 21 days.

163. The compound according to any one of the embodiments, wherein the compound is a GLP-1 receptor agonist as defined in any one of the foregoing embodiments 139-144 and as in the foregoing embodiment 153 PCSK9 inhibitor as defined in any of

164. The compound of embodiment 160, wherein the compound has an apparent EGF (A) Ki (C3) and GLP-1 of up to 5000 (such as up to 4000, such as up to 3000, such as up to 2000, or such as up to 1000) Effectiveness (C1, no HSA) ratio.

165. The compound of embodiment 160, wherein the compound has an apparent EGF (A) Ki (C3) and GLP-1 of up to 1000 (such as up to 800, such as up to 600, such as up to 400 or such as up to 200) Effectiveness (C1, no HSA) ratio.

166. The compound of embodiment 160, wherein the compound has an apparent EGF (A) Ki (C3) and GLP-1 of up to 200 (such as up to 150, such as up to 100, such as up to 50, 25, and 10) Effectiveness (C1, no HSA) ratio.

167. The compound according to any one of the preceding embodiments 137-166, wherein the compound is capable of reducing cholesterol and body weight at least as much as GLP-1 / in vivo in a rat study as described in section C8 herein EGF (A) Compound # 41.

168. The compound of embodiment 167, wherein the compound is capable of reducing cholesterol by at least 0.5 mmol / L when administered at 30 nmol / kg / day and measured after 21 days.

169. The compound of embodiment 167, wherein the compound is capable of reducing cholesterol by at least 0.6, such as 0.7 or such as 0.8 mmol / L when administered at 30 nmol / kg / day and measured after 21 days.

170. The compound of embodiment 167, wherein the compound is capable of reducing cholesterol by at least 0.8 mmol / L when administered at 300 nmol / kg / day and measured after 21 days.

171. The compound of embodiment 167, wherein the compound is capable of reducing cholesterol by at least 1.0 or such as 1.2 mmol / L when administered at 300 nmol / kg / day and measured after 21 days.

172. The compound of embodiments 167-171, wherein the compound is capable of reducing body weight to at least 95% of the baseline BW when administered at 300 nmol / kg / day and measured after 21 days.

173. The compound of embodiments 167-171, wherein the compound is capable of reducing body weight to at least 90% of baseline BW when administered at 300 nmol / kg / day and measured after 21 days.

174. The compound according to any one of the preceding embodiments, wherein the compound is selected from the group of compounds as defined by GLP-1 / EGF (A) compounds # 1 to # 314.

175. A compound comprising a GLP-1 receptor agonist and an EGF (A) analog, wherein the EGF (A) analog is an EGF (A) domain of LDL-R identified by SEQ ID No: 1 ( 293-332).

176. The compound according to embodiment 175, wherein the EGF (A) analog is as defined in any one of the foregoing embodiments 64-89.

177. A compound selected from the group of compounds as defined by GLP-1 / EGF (A) compounds # 1 to # 305.

178. A compound selected from the group of compounds as defined by GLP-1 / EGF (A) compounds # 1 to # 314.

179. A compound selected from the group of compounds defined by: GLP-1 / EGF (A) compound # 1, 2, 21, 22, 23, 25, 26, 27, 29, 32, 41, 48, 51, 52, 53, 54, 69, 82, 86, 221, 230, 287, 298 and 306.

180. A compound selected from the group of compounds defined as: GLP-1 / EGF (A) compound # 1, 2, 21, 22, 23, 25, 26, 27, 29, 32, 48, 52, 53, 54, 69, and 306.

181. A compound selected from the group of compounds defined by, for example, GLP-1 / EGF (A) compounds # 41, # 48, # 69, and # 306 (such as # 306 and # 69, or such as # 306 or # 69) group.

182. The use of a compound according to any one of the preceding embodiments, for use in the manufacture of a pharmaceutical.

183. A compound according to any one of the aforementioned embodiments 1-181, which is used for the preparation of a pharmaceutical.

184. A compound according to any one of the foregoing embodiments 1-181, for use in a method of treatment.

185. A compound according to any one of the foregoing embodiments 1-181, for use in a method of treating diabetes and / or overweight

186. A compound according to any one of the foregoing embodiments 1-181, which is for use in a method of treating or preventing cardiovascular disease and / or cardiovascular risk.

187. A compound according to any one of the foregoing embodiments 1-181, which is for use in a treatment method for improving lipid parameters.

188. A compound according to any one of the aforementioned embodiments 1-181, which is for use in a method of treating diabetes and cardiovascular disease.

189. A method for the treatment of diabetes and / or overweight, the method comprising administering a pharmaceutically active amount of a compound according to any one of the foregoing embodiments 1-181 to a patient in need thereof.

190. A method for the treatment or prevention of cardiovascular disease and / or cardiovascular risk, the method comprising administering a pharmaceutically active amount of a compound according to any one of the foregoing embodiments 1-181 Patient.

191. A method for the treatment of improving lipid parameters, the method comprising administering a pharmaceutically active amount of a compound according to any one of the foregoing embodiments 1-181 to a patient in need thereof.

192. A method for the treatment of diabetes and cardiovascular diseases, the method comprising administering a pharmaceutically active amount of a compound according to any one of the foregoing embodiments 1-181 to a patient in need thereof.

Methods and examples

Abbreviations

Aib: α-aminoisobutyric acid (2-aminoisobutyric acid)

AcOH: acetic acid

Ado: 8-amino-3,6-dioxooctanoic acid

API: Active Pharmaceutical Ingredient

AUC: Area under the curve

BG: Blood glucose

BHK: Hamster Infant Kidney

BW: Weight

Boc: tertiary butoxycarbonyl

BSA: bovine serum albumin

Bzl: benzyl

CAS: Chemical Abstracts Service

Clt: 2-chlorotrityl

Colin base: 2,4,6-trimethylpyridine

DCM: dichloromethane

Dde: 1- (4,4-dimethyl-2,6-dioxocyclohexylene) ethyl

DesH: De-aminohistidine (imidazopropionic acid or 3- (imidazol-5-yl) propionic acid), Imp)

DIC: Diisopropylcarbodiimide

DIPEA: Diisopropylethylamine

DMEM: Dulbecco's Modified Eagle's Medium (DMEM)

DooaSuc: 8-amino-3,6-dioxooctyl succinic acid

DTT: 1,4-dithiothreitol

EDTA: ethylenediamine tetraacetic acid

EGF: epidermal growth factor

EGF (A): Epidermal growth factor domain A

EGTA: ethylene glycol tetraacetic acid

FCS: Fetal Bovine Serum

Fmoc: 9-fluorenylmethoxycarbonyl

HATU: (O- (7-azabenzotriazol-1-yl) -1,1,3,3-tetramethylurenium hexafluorophosphate)

HBTU: (2- (1H-benzotriazol-1-yl-)-1,1,3,3 tetramethylurenium hexafluorophosphate)

HEPES: 4- (2-hydroxyethyl) -1-piperazine Ethanesulfonic acid

HFIP: 1,1,1,3,3,3-hexafluoro-2-propanol or hexafluoroisopropanol

HOAt: 1-hydroxy-7-azabenzotriazole

HOBt: 1-hydroxybenzotriazole

hPCSK9: Human PCSK9

HPLC: High Performance Liquid Chromatography

HSA: human serum albumin

IBMX: 3-isobutyl-1-methylxanthine

IC ₅₀ : half maximum inhibitory concentration

Imp: imidazopropionic acid or 3- (imidazol-5-yl) propionic acid) (also known as deaminated histidine, DesH)

Inp: Isohexahydronicotinic acid

i.v. intravenous

ivDde: 1- (4,4-dimethyl-2,6-dioxocyclohexylene) -3-methylbutyl

IVGTT: intravenous glucose tolerance test

LCMS: liquid chromatography mass spectrometry

LDL-R or LDLr: LDL receptor

LDL: low density lipoprotein

LDL-C: LDL cholesterol

LYD: Landrace Yorkshire Duroc

MALDI-MS: see MALDI-TOF MS

MALDI-TOF MS: Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry

MeOH: methanol

Mmt: 4-methoxytrityl

MRT: average residence time

Mtt: 4-methyltrityl

NMP: N-methylpyrrolidone

ND: Not determined

OBz: benzamidine

OEG: 8-amino-3,6-dioxooctanoic acid (also known as Ado)

OPfp: Pentafluorophenoxy

OPnp: p-nitrophenoxy

OSu: O-succinimide (hydroxysuccinimide)

OtBu: tertiary butyl ester

Oxyma Pure®: cyano-hydroxyimino-ethyl acetate

Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl

PBS: phosphate buffered saline

PD: Pharmacodynamics

Pen / Strep: Penicillin / Streptomycin

PK: Pharmacokinetics

QC: Quality Control

RP: reverse phase

RP-HPLC: Reverse Phase High Performance Liquid Chromatography

RT: room temperature

Rt: residence time

s.c .: subcutaneous

SD: standard deviation

SEC-HPLC: High Performance Liquid Chromatography with Particle Size Sieve Analysis

SEM: mean standard error

SPA: Proximity Flicker Analysis

SPPS: Solid Phase Peptide Synthesis

tBu: tertiary butyl

TFA: trifluoroacetic acid

TIS or TIPS: Triisopropylsilane

Tos: Tosylate (or p-toluenesulfonyl)

TotaGlyc: 13-amino-4,7,10-trioxotridecyldiethylene glycol diglycolamic acid

Tris: Tris (hydroxymethyl) aminomethane or 2-amino-2-hydroxymethyl-propan-1,3-diol

Trt: triphenylmethyl (trityl)

Trx: Tranexamic acid

TtdSuc: 13-amino-4,7,10-trioxotridecylsuccinic acid

UPLC: Ultra High Performance Liquid Chromatography

special material

Eicosanedioic acid mono-tertiary butyl ester

Dodecanedioic acid mono-tertiary butyl ester

Tert-Butyl 4- (10-carboxydecyloxy) benzoate

Fmoc-8-amino-3,6-dioxooctanoic acid

Fmoc-tranexamic acid

Fmoc-Lys (Mtt) -OH

Boc-His (Trt) -OH

Fmoc-Aib-OH

The preparation of eicosanedioic acid mono-tertiary butyl ester, behenedioic acid mono-tertiary butyl ester, and 4- (10-carboxydecoxy) benzoic acid tertiary butyl ester is described in Section 2 below, The last five mentioned materials are commercially available.

method

This chapter is divided into three sections: Section A is about the general method for the preparation of compounds of the invention, Section B is about the preparation of some special compounds of the invention, and Section C is about the methods of defining characteristics of the compounds of the invention (Also includes the results of some specific example compounds).

A1. General method of preparation

This chapter is about methods for solid phase peptide synthesis (SPPS methods, including methods for deprotection of amino acids, methods for shearing peptides from resins, and their purification), and Methods for detecting and defining the characteristics of the resulting peptides (LCMS and UPLC methods).

In some examples, the solid-phase synthesis of peptides can be accomplished by diacid amido bonds with cleavable groups under acidic conditions (such as, but not limited to, 2-Fmoc-oxy-4-methoxy Use of benzyl, or 2,4,6-trimethoxybenzyl) protected dipeptides to improve. In examples where serine or hydroxybutyric acid is present in the peptide, a pseudoproline dipeptide (available from, for example, Novabiochem, see also WRSampson (1999), J. Pep. Sci .5,403). Fmoc-protected amino acid derivatives are recommended standards: Fmoc-Ala-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Asp (OtBu)- OH, Fmoc-Cys (Trt) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Gly-OH, Fmoc-His (Trt) -OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys (Boc) -OH, Fmoc-Met-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH Fmoc-Trp (Boc) -OH, Fmoc-Tyr (tBu) -OH, or Fmoc-Val-OH, etc., supplied by, for example, Anaspec, Bachem, Iris Biotech, or Novabiochem. When not otherwise specified, the natural L form of amino acids is used. The N-terminal amino acid is a Boc-protected α-amino group (for example, a peptide having His at the N-terminus is Boc-His (Boc) -OH, or Boc-His (Trt) -OH). Examples of attachment of a modular albumin binding moiety using SPPS, such as, but not limited to, Fmoc-8-amino-3,6-dioxocaprylic acid, Fmoc-tranexamic acid, Fmoc-isohexanicotin The following suitably protected building blocks of acids, Fmoc-Glu-OtBu and cetyl monotert-butyl ester are supplied by, for example, Anaspec, Bachem, Iris Biotech, or Novabiochem. Eicosanedioic acid monotertiary butyl ester, behenedioic acid monotertiary butyl ester, and 4- (10-carboxydecyloxy) benzoic acid tertiary butyl ester can be prepared as described below. All operations described below were performed on a 400-μmol or 450-μmol synthesis scale.

    1. Synthesis of protected peptide hydrazine bound to resin     Method: SPPS_P

SPPS_P is a 400-μmol or 450-μmol scale used on a Prelude or SymphonyX solid-phase peptide synthesizer from Protein Technologies (Tucson, AZ 85714 USA) compared to resin loading (e.g., Fmoc- Hydrazine-pyruvyl-aminomethyl polystyrene resin (PYV1000, from Iris Biotech, 95615 Marktwick, Germany)) A five-fold excess of Fmoc-amino acid (300 mM in DMF) , Plus 300mM Oxyma Pure®). Fmoc-deprotection was performed using 20% piperidine in DMF or 20% piperidine in DMF plus 0.1M Oxyma Pure®. Coupling was performed using 5: 5: 5: 5 Amino Acid / Oxyma Pure® / DIC / Collin in DMF. The DMF top wash (6 cycles of 9 ml) was performed between the deprotection and coupling steps. Coupling time is generally 120 minutes. Some amino acids (including, but not limited to) Fmoc-Arg (Pbf) -OH, Fmoc-Aib-OH, or Boc-His (Trt) -OH) are "double coupled", meaning the first coupling (e.g. ), Let the resin dry and add more reagents (amino acid, Oxyma Pure®, DIC, and colin base) and allow the mixture to react again (for example, 60 min).

    2. Synthesis of protected Cys-peptidic acid combined with resin    

SPPS_P was performed using the same coupling procedure using the low loading Fmoc-Glu (OtBu) -Wang (0.32 mmol / g) resin as described above.

    3. Synthesis of albumin binder    

Eicosanedioic acid mono-tert-butyl ester can be prepared as known in the art, for example as described in WO 2010102886 A1.

The dodecanedioic acid mono-tertiary butyl ester can be prepared as known in the art, for example as described in WO2015000942 A1.

Tert-butyl 4- (10-carboxydecoxy) benzoate can be prepared as known in the art, for example as described in WO2006082204 A1

    4. Attachment of side chain to resin-protected peptide backbone    

When the tritiated system is present on the side chain of the lysine, the epsilon amino group of the lysine to be tritiated is protected by Mtt. The removal of Mtt was performed using hexafluoroisopropanol / DCM (75:25, 3 x 10ml, 5min, 25min, and 25min respectively), followed by DCM (4x 10ml), DMF (2x 9ml), and 20% in DMF Piperidine plus 0.1M Oxyma Pure® (1x 9ml), DMF (4x 9ml) washing resin. The elongation moiety and / or linker can be attached to the peptide by tritiation of the resin-bound peptide or by tritiation in a solution of the unprotected peptide (as described in WO2010029159 A1). . In the example of attachment of an elongation moiety and / or a linker to a protected peptide-based resin, the attachment may be modularized using SPPS and suitably protected construction elements.

Method: SC_P

The N-ε-lysine protecting group is removed as described above and the chemistry of the lysine is performed on a Prelude or SymphonyX peptide synthesizer by one or more automated steps using a suitable protected building block as described above Modification. A double coupling (1 hour per coupling) or a single coupling (2 hours per coupling) was performed as described in SPPS_P.

    5.Cleaving and purification of peptides bound to resin with or without attached side chains     Method: CP_M1

After synthesis, the resin was washed with DCM, and the peptide was sheared from the resin by treating with TFA / TIS / water / DTT (92.5 / 2.5 / 2.5 / 2.5 or 90/5 / 2.5 / 2.5) for 2-3 hours This was followed by precipitation with diethyl ether. The peptide was dissolved in a suitable solvent such as water / acetonitrile and purified by standard RP-HPLC on a C18, 5 μm column using acetonitrile / water / TFA. Fractions were analyzed by a combination of UPLC and LCMS methods, and appropriate fractions were poured together and freeze-dried.

If desired, the peptide can be relatively ion-exchanged to sodium using methods known in the art. As an example, a 5gram Sep-pak C18 column was washed with 50 ml of 2-propanol, 50 ml of acetonitrile, and 50 ml of water. A solution of approximately 70 mg of protein in 21 ml of 50 mM HEPES-buffer (pH 7.2) was loaded onto a Sep-pak column, which was washed with 50 ml of water, 50 ml of 0.1 M sodium chloride (aq), and 50 ml of water. The sodium salt of the protein was eluted with 100 ml of water / acetonitrile (30:70) and freeze-dried.

    6. Natural chemical ligation and purification of peptide hydrazine using Cys-peptide     Method: NCL_M1

The peptide hydrazine (1.0 eq) was dissolved in 0.2 M disodium phosphate / 6.0 M guanidine hydrochloride (aq, pH 3.0) to a final concentration of 4.0 Mm and cooled to -10 ° C. Sodium nitrite (0.2M in water, 5eq) was added, and the mixture was stirred at -10 ° C for 20 minutes. A solution of 0.2M 4-mercaptophenylacetic acid (50eq) in 0.2M disodium phosphate / 6.0M guanidine hydrochloride (pH adjusted to 7.0) was added to the above solution, followed by Cys-peptide (1.1eq). . The pH of the solution was adjusted to 6.7 using sodium hydroxide (1.0M, aq) and the mixture was stirred at 25 ° C for 16 hours. 1,4-Dithiothreitol (100 eq) was added to the reaction mixture and stirred for 30 minutes, after which the pH was adjusted to 3.0 with concentrated hydrochloric acid (aq). The reaction mixture was concentrated by ultrafiltration using an Amicon Ultra-15 centrifugal filtration unit using an Ultracel-3 membrane from EMD Millipore (Billerica, MA 01821 U.S.A.). The concentrated solution was diluted with 0.05M disodium phosphate / 6.0M guanidine hydrochloride (aq, pH 3.0) and concentrated by ultrafiltration again. This was repeated until the concentration of 4-mercaptophenylacetic acid was below 0.1 mM (which corresponds to a> 1000-fold dilution). The concentrated solution was added dropwise to a stirred solution of 50 mM tris (hydroxymethyl) aminomethane, 5 mM calcium chloride, 3 mM cysteine, 0.3 mM cystine, (aq, pH 8.2) to obtain app .0.1 mg / ml protein concentration. The solution was stirred at 25 ° C for 16 hours. The pH of the folded mixture was adjusted to app. 3 with concentrated hydrochloric acid (aq), and then purified by standard RP-HPLC on a C18, 5 μm column using acetonitrile / water / TFA. Fractions were analyzed by a combination of UPLC and LCMS methods, and appropriate fractions were poured together and freeze-dried.

    7. Recombinant performance of fusion proteins    

Target fusion proteins are provided by heterologous expression using a suitable host. The phenotypic system is constructed using known techniques and the fusion protein is expressed and purified by methods known to those skilled in the art. Briefly, cells were harvested and lysed in a 1X PBS buffer at pH 7 by a cell breaker. The insoluble fraction (which contains the fusion protein) was collected and washed twice in the same buffer (6,000 g / 20 min). Then, at room temperature (22-26 ° C), 20 mM ethanolamine, 2M urea, and pH 10.5 were used to dissolve the inclusion bodies to a concentration of 10 mg / mL. After one hour, the solution was diluted 3 times with demineralized water and the pH was adjusted to 8.5. Enterokinase cleavage was performed at a ratio of 1: 1,000 for 20 hours at the same temperature. After that, CaCl ₂ and 5 mM cysteine were added in a final concentration to refold. After the pH was adjusted to 3.0, the protein was captured from SP rapid flow sepharose. The captured sample was applied to a reverse-phase FeF column at pH 7.5. A Source 30Q column (20 mM Tris, 5 mM CaCl ₂ , pH 9.0) was selected as the final processing step.

    8. Incorporation of non-proteinogenic amino acids in recombinant proteins    

The N-terminal His-Aib dipeptide can be introduced by dehydration with Fmoc-His-Aib-OH in solution followed by removal of the Fmoc-protecting group, as described in WO2013098191 A1.

A2. General methods for detecting and defining features     LC-MS method     Method: LCMS01

LCMS01 was performed on a setup consisting of a Waters Acquity UPLC system and an LCT Premier XE mass spectrometer from Micromass. Eluent: A: 0.1% formic acid in water; B: 0.1% formic acid in acetonitrile. The analysis is performed at RT by injecting an appropriate volume (preferably 2-10 μl) of a sample onto a column (which is a gradient dissolution of A and B). UPLC conditions, detector settings, and mass spectrometer settings: Column: Waters Acquity UPLC BEH, C-18, 1.7 μm, 2.1 mm x 50 mm. Gradient: linear 5% -95% acetonitrile at 0.4ml / min during 4.0min (optionally 8.0min). Detection: 214nm (analog output from TUV (tunable UV detector)) MS ionization mode: API-ES. Scan: 100-2000amu (alternatively 500-2000amu), step 0.1amu.

Method: LCMS34

LCMS34 was performed on a setup consisting of a Waters Acquity UPLC system and a Xevo G2-XS Qtof mass spectrometer. Eluent: A: 0.1% formic acid in water; B: 0.1% formic acid in acetonitrile. The analysis is performed at RT by injecting an appropriate volume (preferably 2-10 μl) of a sample onto a column (which is a gradient dissolution of A and B). UPLC conditions, detector settings, and mass spectrometer settings: Column: Waters Acquity UPLC BEH, C-18, 1.7 μm, 2.1 mm x 50 mm. Gradient: linear 5% -95% acetonitrile at 0.4ml / min during 4.0min (optionally 8.0min). Detection: 214nm (analog output from TUV (tunable UV detector)) MS ionization mode: API-ES. Scan: 100-2000amu (alternatively 500-2000amu), step 0.1amu.

Method: LCMS27

The LCMS27 is based on an Agilent 1290 Infinity Series and an Agilent Technologies LC / MSD TOF 6230 (G6230A) detector (with Agilent Jet Stream source ionization). Performed on the composed settings. Eluate: A: 0.02% TFA in water; B: 0.02% TFA in acetonitrile. The analysis is performed at RT by injecting an appropriate volume (preferably 2-10 μl) of a sample onto a column (which is a gradient dissolution of A and B). UPLC conditions, detector settings, and mass spectrometer settings: Column: Aeris Widepore, C-18, 3.6 μm, 2.1 mm x 50 mm. Gradient: linear 5% -95% acetonitrile over a period of 4.0 min at 0.4 ml / min. Detection: 214nm (analog output from TUV (tunable UV detector)). Scanning: 100-3200amu.

    2. UPLC method     Method: UPLC01

RP-analysis was performed using a Waters UPLC system equipped with a dual-band detector. UV detection at 214nm and 254nm was collected using ACQUITY UPLC BEH130, C18, 130Å, 1.7um, 2.1mm x 150mm column (40 ° C). The UPLC system is connected to two eluent reservoirs containing: A: 99.95% H ₂ O, 0.05% TFA; B: 99.95% CH ₃ CN, 0.05% TFA. The following linear gradients were used: 95% A, 5% B to 40% A, 60% B, at a flow rate of 0.40 ml / min over a period of 16 minutes.

Method: UPLC02

RP-analysis was performed using a Waters UPLC system equipped with a dual-band detector. UV detection at 214nm and 254nm was collected using ACQUITY UPLC BEH130, C18, 130Å, 1.7um, 2.1mm x 150mm column (40 ° C). The UPLC system is connected to two eluent reservoirs containing: A: 99.95% H ₂ O, 0.05% TFA; B: 99.95% CH ₃ CN, 0.05% TFA. The following linear gradients were used: 95% A, 5% B to 5% A, 95% B, at a flow rate of 0.40 ml / min over a period of 16 minutes.

A3. Definition of characteristics of selected intermediates

Peptide hydrazine:

[8Aib, 34R] GLP-1 (7-37) -GQAPGQAP- [301L] EGF (A) (293-303) Hydrazine, with 26K (of ε nitrogen) attached substituent # 1 (HOOC- (CH ₂ ) ₁₆ -CO-γGlu-Ado-Ado).

Preparation method: SPPS_P; CP_M1

LCMS34: m / 3 = 1970.3, m / 4 = 1478.0, m / 5 = 1182.6

UPLC02: Rt = 9.3min

Cys-peptide:

[309R, 312E, 321E] EGF (A) (304-332)

Preparation method: SPPS_P; CP_M1

LCMS01: m / 3 = 1119.8, m / 4 = 840.1, m / 5 = 672.3

UPLC02: Rt = 8.4min

B1. Special compounds-EGF (A) analogs and derivatives

Summary Table of EGF (A) Analogs and Derivatives (EGF (A) Compound 1-159)

B2. Special compound-GLP-1 / EGF (A) compound

Preparation of the compounds is performed as described above. Why is the compound provided by reference to the amino acid sequence of each element, its substituents, and the particular attachment points of one or two substituents as provided elsewhere in this document. Some examples are shown below and summary tables are provided below.

GLP-1 / EGF (A) Compound # 1

It can also be described as: [8Aib, 34R] GLP-1 (7-37) -GQAPGQAP- [301L, 309R, 312E, 321E] EGF (A), which has passed -37) of 26K (εε nitrogen) attached substituent # 1 . Or [8Aib, 34R] GLP-1 (7-37) -GQAPGQAP- [301L, 309R, 312E, 321E] EGF (A), which has 26K ( (Ε-nitrogen) attached substituent # 1 (HOOC- (CH ₂ ) ₁₆ -CO-γGlu-Ado-Ado). Or SEQ ID 193, which has a substituent attached via a 26K (ε nitrogen) of an lysine (K) (equal to [8Aib, 34R] GLP-1 (7-37)) at position 20 of SEQ ID 193 # 1.

GLP-1 / EGF (A) Compound # 23

It can also be described as: [8Aib, 34R] GLP-1 (7-37) -GQAPGQAP- [301L, 309R, 312E, 321E] EGF (A), which has passed [8Aib, 34R] GLP-1 (7 -37) of 26K (εε nitrogen) attached substituent # 6 . Or [8Aib, 34R] GLP-1 (7-37) -GQAPGQAP- [301L, 309R, 312E, 321E] EGF (A), which has passed [8Aib, 34R] GLP-1 (26-K of 7-37 (of ε nitrogen) attached substituent # 6 (HOOC- (CH ₂ ) ₁₈ -CO-Trx-γGlu-Ado-Ado). or SEQ ID 193, which has an lysine through (position 20 of SEQ ID 193 K) Attached substituent # 6.

GLP-1 / EGF (A) Compound # 41

It can also be described as [8Aib, 34R] GLP-1 (7-37) -GQAPGQAP- [301L, 309R, 312E, 321E] EGF (A), which has passed [8Aib, 34R] GLP-1 26K (of ε nitrogen) and [301L, 309R, 312E, 321E, 333K] 333K (ε nitrogen) attached to EGF (A) # 1 Or [8Aib, 34R] GLP-1 (7-37) -GQAPGQAP- [301L, 309R, 312E, 321E, 333K] EGF (A), which has passed [8Aib, 34R] GLP-1 26K (εε nitrogen) And [301L, 309R, 312E, 321E, 333K] Substituent # 1 (HOOC- (CH ₂ ) ₁₆ -CO-γGlu-Ado-Ado) or SEQ ID attached to 333K (ε nitrogen) of EGF (A) 190, which has a substituent # 1 attached by [8Aib, 34R] GLP-1 26K (ε nitrogen) and [301L, 309R, 312E, 321E, 333K] EGF (A) 333K (ε nitrogen) HOOC- (CH ₂ ) ₁₆ -CO-γGlu-Ado-Ado). Or SEQ ID 190, which has a substituent # 1 (HOOC- (CH ₂ ) ₁₆ -CO-γGlu-Ado-Ado) attached via Lys at positions 20 and 80.

GLP-1 / EGF (A) Compound # 42

It can be described as [8Aib, 26R, 34R] GLP-1 (7-37) -GQAPGQAP- [301L, 309R, 312E, 313K, 321K] EGF (A), which has passed through [301L, 309R, 312E, 313K 321K] 313K and 321K (ε nitrogen) attached substituents of EGF (A) # 13 . Or [8Aib, 26R, 34R] GLP-1 (7-37) -GQAPGQAP- [301L, 309R, 312E, 313K, 321K] EGF (A), which has passed [301L, 309R, 312E, 313K, 321K] EGF (A) 313K and 321K (ε nitrogen) attached substituent # 13 (4-COOH-PhO-C11-γGlu-Ado-Ado). Or SEQ ID 380, which has a substituent # 13 (4-COOH-PhO-C11-γGlu- Ado-Ado). Or SEQ ID 380, which has a substituent # 13 attached via positions 60 and 68 Lys at SEQ ID 380.

GLP-1 / EGF (A) Compound # 75

It can also be described as [301L, 309R, 312E, 321E] EGF (A) -GQAPGQAP- [8Aib, 34R] GLP-1 (7-37), which has the 37) of 26K (ε nitrogen) attached substituent # 1 Or [301L, 309R, 312E, 321E] EGF (A) -GQAPGQAP- [8Aib, 34R] GLP-1 (7-37), which has 26K ( (Ε-nitrogen) attached substituent # 1 (HOOC- (CH ₂ ) ₁₆ -CO-γGlu-Ado-Ado) or SEQ ID 386, which has a structure of [8Aib, 34R] GLP-1 (7-37) 26K (ε nitrogen) attached substituent # 1 (HOOC- (CH ₂ ) ₁₆ -CO-γGlu-Ado-Ado). Or SEQ ID 386, which has a substituent # 1 attached via Lys at position 68 of SEQ ID 386. (7-37).

Why are further compounds provided by reference to the amino acid sequence of each element (as provided elsewhere herein), its substituents, and special attachment points for one or two of its substituents.

    Summary table containing GLP-1 analogs and derivatives of EGF (A) analogs (GLP-1 / EGF (A) compounds)    

The attachment of its substituents is indicated by reference to its GLP-1 and EGF (A) analogs, respectively. As noted above, 26K is equal to position 20 in the GLP-1 (7-37) sequence and 324K of the EGF (A) analog is located at position 32 of the EGF (A) domain (293-332) analog of LDL-R Amine substitution. Special locations for other attachment locations can be inferred in a similar manner. The particular position of the (and other) substituents relative to the peptide backbone will vary based on the spacers of GLP-1 and EGF (A) analogs and the possible truncation of length.

Compound with EGF (A) analog at the C-terminus

Compounds with an EGF (A) analog at the N-terminus

The analytical data used for compound selection are provided in the table below.

Table containing analytical data for GLP-1 / EGF (A) compounds

C. General methods for defining characteristics

To define the characteristics of these compounds, their functionality can be tested in various analyses.

    C1-GLP-1 in vitro efficacy    

The purpose of this analysis is to test the GLP-1 activity (or potency) of a compound, such as a derivative comprising a GLP-1 analog, in a test tube. In-vitro potency is a measure of human GLP-1 receptor activation in a whole-cell assay.

The potency of the derivatives of the GLP-1 / EGF (A) compound was determined as described below and included data for GLP-1 (7-37) and Soma Valley peptides for comparison.

    Principle    

In-vitro potency was determined by measuring the response of the human GLP-1 receptor in a reporter gene analysis. Analysis of stably transfected BHK cell lines expressing human GLP-1 receptor and containing cAMP response element (CRE) (CRE luciferase) DNA linked to promoter and gene of firefly luciferase carried out. When the human GLP-1 receptor is activated, it results in the production of cAMP, which in turn causes the luciferase protein to be expressed. When the analytical culture is completed, a luciferase substrate (luciferin) is added and the enzyme converts luciferin to oxidized luciferin to produce bioluminescence. Luminescence is measured as an analytical readout.

    Cell culture and preparation    

The cells used for this analysis (asexual propagation line FCW467-12A / KZ10-1) were BHK cells with BHKTS13 as the parental cell line. These cell lines are derived from a clonal propagation line (FCW467-12A) expressing the human GLP-1 receptor and are established by further transfection with CRE luciferase to obtain the current clonal propagation line.

The cells were cultured in a cell culture medium under 5% CO ₂ . It was aliquoted and stored in liquid nitrogen. Prior to each analysis, aliquots were taken and washed twice in PBS, and then suspended in the analysis-specific buffer at the desired concentration. For 96-well plates, suspensions were made to give a final concentration of 5x10 ³ cells / well.

    Material    

The following chemicals were used in the analysis: Pluronic F-68 (10%) (Gibco 2404), human serum albumin (HSA) (Sigma A9511), ovalbumin (Sigma A5503), phenol-free DMEM (Gibco 11880-028) , 1M Hepes (Gibco 15630), Glutamax 100x (Gibco 35050) and steadylite plus (PerkinElmer 6016757).

    Buffer    

The cell culture medium is a DMEM medium with 10% FBS (fetal bovine serum; Invitrogen 16140-071), 1 mg / ml G418 (Invitrogen 15140-122), 240 nM MTX (methoxamine; Sigma M9929), and 1% pen / strep ( Penicillin / Streptomycin; Invitrogen 15140-122).

The analysis medium was phenol red-free DMEM, 10 mM Hepes, and 1 x Glutamax. The analysis buffer is composed of 2% ovalbumin and 0.2% Pluronic F-68 in the analysis medium.

    Procedure    

1) Thaw the cell stock in a 37 ° C water bath.

2) Wash the cells three times in PBS.

3) Count the cells and adjust to 5x10 ³ cells / 50 μl (1x10 ⁵ cells / ml) in the analysis medium. A 50 μl aliquot of the cells was transferred to each well in the analysis plate.

4) Dilute stocks of test compounds and reference compounds in assay buffer to a concentration of 0.2 μM. Compounds were diluted 10-fold to give the following concentrations: 2x10 ^-7 M, 2x10 ^-8 M; 2x10 ^-9 M, 2x10 ^-10 M, 2x10 ^-11 M, 2x10 ^-12 M, 2x10 ^-13 M, and 2x10 ^-14 M.

5) Transfer 50 μl aliquots of the compound or blank from the dilution plate to the analysis plate. The compounds were tested at the following final concentrations: 1x10 ^-7 M, 1x10 ^-8 M; 1x10 ^-9 M, 1x10 ^-10 M, 1x10 ^-11 M, 1x10 ^-12 M, 1x10 ^-13 M, and 1x10 ^-14 M.

6) The analysis plate was cultured at 37 ° C for 3 h in a 5% CO ₂ incubator.

7) Remove the analysis plate from the incubator and allow it to stand at room temperature for 15 min.

8) Add 100 μl aliquots of steadylite plus reagents to each well of the analysis plate (reagents are photosensitive).

9) Cover each analysis plate with aluminum foil to protect it from light and shake at room temperature for 30 min.

10) Read each analysis disk in a Packard TopCount NXT instrument.

    Calculations and results    

In-vitro potency analysis as described above was performed on a range of compounds, including and not including HSA. Transfer data from TopCount instruments to GraphPad Prism software. This software performs non-linear regression (log (agonist) vs response). The EC ₅₀ values calculated by this software and reported in pM are shown in Table 1 below.

For each sample, measure at least two replicates. Reported values are averages of duplicates.

Most GLP-1 / EGF (A) compounds show GLP-1 activity. The particular effectiveness (in the absence and presence of HSA) is affected by amino acid changes and spacers and substituents in the analogs. The above data show that compounds with comparable or reduced potency compared to GLP-1 (7-37) and Soma Valley such as peptides can be obtained.

In addition, when the EGF (A) analog was attached to the N-terminus of the GLP-1 analog (Compound 75, SEQ ID 386) rather than the C-terminus of the GLP-1 analog (Compound 1, SEQ ID 193), observe To a significant loss of GLP-1 efficacy.

    C2-GLP-1- in vitro receptor binding    

The purpose of this example is to test the receptor binding of GLP-1 derivatives in a test tube. A measure of the affinity of a receptor-binding derivative for the human GLP-1 receptor.

    Principle    

Receptor binding to the human GLP-1 receptor was measured in a competitive binding assay. In this type of assay, the labeled ligand ( ¹²⁵ I-GLP-1 in this example) is bound to its receptor. Each derivative / compound was added to the isolated human GLP-1 receptor-containing membrane at a series of concentrations and the replacement of the labeled ligand was monitored. Receptor binding is reported as the concentration (IC ₅₀ value) at which one-half of its labeled ligand is displaced from the receptor. GLP-1 (7-37) and Somaglutinin are included as comparative compounds.

    Material    

The following chemicals were used in this analysis: human serum albumin (HSA) (Sigma A1653), phenol red-free DMEM (Gibco 11880-028), Pen / strep (Invitrogen 15140-122), G418 (Invitrogen 10131-027), 1M Hepes (Gibco 15630), EDTA (Invitrogen 15575-038), PBS (Invitrogen 14190-094), fetal bovine serum (Invitrogen 16140-071), EGTA, MgCl ₂ (Merck 1.05832.1000), Tween 20 (Amresco 0850C335) SPA particles (wheat germ agglutinin (WGA) SPA beads, Perkin Elmer RPNQ0001), [ ¹²⁵ I] -GLP-1]-(7-36) NH ₂ (home-made), OptiPlate ^TM -96 (Packard 6005290).

Buffer 1 was composed of 20 mM Na-HEPES plus 10 mM EDTA and the pH was adjusted to 7.4. Buffer 2 was composed of 20 mM Na-HEPES plus 0.1 mM EDTA and the pH was adjusted to 7.4. The analysis buffer was composed of 50 mM HEPES supplemented with 5 mM EGTA, 5 mM MgCl ₂ and 0.005% Tween 20, and the pH was adjusted to 7.4. The 8% albumin stock solution was dissolved in 8% (w / v) in the analysis buffer. The composition of HSA in the agent. The 0.02% albumin stock is composed of HSA dissolved in analysis buffer at 0.02% (w / v).

    Cell culture and membrane preparation    

The cells used for this analysis (asexual propagation line FCW467-12A) were BHK cells with BHKTS13 as the parental cell line. These cells express the human GLP-1 receptor.

The cell line was grown in DMEM, 10% fetal bovine serum, 1% Pen / Strep (penicillin / streptomycin) and 1.0 mg / ml of the selection marker G418 under 5% CO ₂ . To make a membrane preparation, cells were grown to approximately 80% confluence. Cells were washed twice in phosphate buffered saline and harvested. Cells were pelleted into pellets using brief centrifugation and the cell pellets were placed on ice. Cell pellets are homogenized in an appropriate amount of buffer 1 (e.g., 10 ml) with a ULTRA-THURRAX ^™ disperser for 20-30 seconds. Centrifuge the homogenate for 15 minutes. The pellets were resuspended (homogenized) in 10 ml of Buffer 2 and centrifuged. Repeat this step once. The resulting pellets were resuspended in buffer 2 and the protein concentration was measured. The film was divided into aliquots and stored at minus 80 ° C.

    Procedure    

1) For receptor binding analysis in the presence of low HSA (0.005%), add 50 μl of analysis buffer to each well of the analysis plate.

2) The test compound was serially diluted to give the following concentrations: 8x10 ^-7 M, 8x10 ^-8 M, 8x10 ^-9 M, 8x10 ^-10 M, 8x10 ^-11 M, 8x10 ^-12 M, and 8x10 ^-13 M. Twenty-five [mu] l was added to a suitable slot in the analysis plate.

3) Melt and dilute the cell membrane aliquots to their working concentrations. Fifty μl was added to each well in the analysis plate.

4) WGA SPA beads were suspended at 20 mg / ml in analysis buffer. The suspension was diluted to 10 mg / ml in analysis buffer and immediately added to the analysis plate. Fifty μl was added to each well in the analysis plate.

5) The culture system by 25μl of ^{[125 I] -GLP-1]} - (7-36) NH 2 was added to 480pM of each of the assay plate starting slot. A 25 μl aliquot was reserved for measuring total counts / wells.

6) Incubate the analysis plate at 30 ° C for 2h.

7) Centrifuge the analysis plate for 10 min.

8) Read the analysis disk in a Packard TopCount NXT instrument.

    Calculation    

Transfer data from TopCount instruments to GraphPad Prism software. This software performs non-linear regression. IC ₅₀ values are calculated by this software and reported in nM.

    The result    

Get the following results:

The above data shows that GLP-1 binding depends on specific sequences and substituents and can obtain various levels of GLP-1 binding activity to prepare compounds that are comparable or reduced compared to GLP-1 (7-37) or Soma Valley such as peptides. Receptor-bound compounds. Again, when the EGF (A) analog is attached to the N-terminus of the GLP-1 analog (Compound 75, SEQ ID 386) instead of the C-terminus of the GLP-1 analog (Compound 1, SEQ ID 193), A significant loss of GLP-1 binding was observed.

    C3-PCSK9-LDL-R binding-competitive (ELISA)    

This analysis measures the apparent binding affinity for PCSK9 competing with LDL-R. Specifically, this analysis was used to assess the apparent binding affinity of EGF (A) analogs and compounds containing EGF (A) analogs, such as GLP-1 / EGF (A) compounds, to PCSK9.

This analysis is performed as follows. On the day before the experiment, the recombinant human low density lipoprotein receptor (rhLDL-R; NSO-derived; R & D Systems # 2148-LD) was dissolved in 50 mM sodium carbonate, pH 9.6 at 1 μg / ml, and then 100 μl of this solution was added to each well of an analysis plate (Maxisorp 96, NUNC # 439454) and coated overnight at 4 ° C. On the day of the experiment, an 8-point concentration curve of the EGF (A) compound containing biotinylated PCSK9 (0.5ug / ml, BioSite / BPSBioscience cat # 71304) was made in duplicate. Test compounds and biotinylated PCSK9 mixtures were prepared and incubated for 1 hour at room temperature in an analysis buffer containing: 25 mM Hepes, pH 7.2 (15630-056, 100 ml, 1 M), 150 mM NaCl (Emsure 1.06404.1000 ) 1% HSA (Sigma A1887-25G) 0.05% Tween 20 (Calbiochem 655205) 2 mM CaCl ₂ (Sigma 223506-500G). The coated analysis disk was then washed 4x in 200 μl of analysis buffer, and then 100 μl of a mixture of the test compound and biotinylated PCSK9 was added to the plate and incubated at room temperature for 2 h. The plates were washed 4x in 200 μl of analysis buffer and then incubated with streptavidin-HRP (25ng / ml; VWR # 14-30-00) for 1 h at room temperature. The reaction was detected by adding 50 μl of TMB-on (KEM-EN-TEC) and incubating in the dark for 10 min. The reaction was then stopped by adding 50 μl of 4M H ₃ PO ₄ to the mixture (by using an electronic multiplex pipette). The plates were then tied to Spectramax and read at 450 and 620 nm in 1 h. The 620nm reading was used for background subtraction. The IC50 value is calculated using Graphpad Prism by non-linear regression log (inhibitor) vs. response-change slope (four parameters) and converted to Ki value using the following formula: Ki = IC50 / (1+ (Biotin- PCSK9) / (kd (Biotin-PCSK9))), wherein the Kd of biotin-PCSK9 is 1.096727714 μg / ml and [Biotin-PCSK9] = 0.5 (μg / ml).

The results are shown in Tables 3.1 to 3.6 below. A higher Ki value reflects a lower apparent binding affinity for PCSK9 and vice versa. Note that several of these compounds showed a Ki that was substantially higher than the values measured for EGF66, such as values above 500 nM, indicating that the observed binding was not specific. The amino acid substitution of the peptide and / or derivatization of one or more side chains can contribute to the loss of binding to LDL-R. All in all, a large number of the EGF (A) compounds tested showed the ability to inhibit the binding of PCSK9 to hLDL-R.

    PCSK9 inhibitor    

Initially, a group including various amino acid substituted EGF (A) analogs was tested as described above and the results are shown in Table 3.1.

EGF66 (EGF (A) compound # 48), which was identified as the most effective peptide variant in WO 2012177741, had 5 mutations. Those who found these mutations were of little importance for the Ki values determined in the analysis described in C3. Specifically, the compound including the wild-type residue Asp (D) at position 310 was found to be more potent than the compound having 310K. In addition, it appears that the key amine substitution system is 301L, preferably in combination with 309R. Finally, 307I and 299A did not contribute much to the affinity of these EGF (A) analogs.

    N-terminal attachment of substituents    

In subsequent experiments, it was tested whether the attachment of half-life extenders (e.g., substituents) to the peptides would affect Ki as determined by the analysis described in C3. As described herein, the substituents may be attached by different techniques and the substituents were originally attached to the nitrogen atom of the N-terminal amino acid of the peptides by halogenation or alkylation.

As can be seen in Table 3.2, all tested compounds have Ki values below 3.0 nM, meaning that these various extender and linker elements are well tolerated. This is unusual because potency is often negatively affected by attachment of side chains, as previously observed for peptides such as GLP-1.

    Lys attachment of substituents    

To evaluate alternative positions for the attachment of substituents to PCSK9 inhibitor peptides, a series of compounds were prepared. A backbone peptide including three amino acid substitutions (N301L, N309R, and K312E) was used, except for EGF (A) compounds # 58, 29, and 4 in combination with Lys substitutions at various positions. All compounds tested included 6 cysteine amino acids at positions 297, 304, 308, 317, 319, 331, which are usually joined in cysteine disulfide bonds. 312E is included to ensure position-specific substitutions, except for EGF (A) compound # 4 (wherein attachment to wt 312K is obtained). Extension of the peptide with one Lys (EGF (A) compounds 75 and 3) was also tested. The substituents described above including the C18 diacid extender and the γGlu-2xAdo linker were used in all compounds and were attached by tritiation. The results are contained in Table 3.3.

This analysis shows that most of the PCSK9 inhibitor peptides maintain functionality. Exceptions are Lys substitutions and derivatives at any of positions 298, 301, 302, and 307, which result in non-functional peptides. It was also observed that the introduction and substitution of Lys at positions 296, 299, 315, and 320K reduced apparent affinity.

These data therefore also confirm the results from Table 3.1, stating that the substitution of Asn (N) 301 with the amino acid of Leu (L) is necessary for binding.

No data was observed for Lys introduction and substitution at positions 295 and 310. As described above, the maintenance of Asp which has been previously found at 310 is better than that of 310K substitution. As can be seen below, the binding was also found to be eliminated by the introduction of Asp (D) (EGF (A) Example Compound 70) at position 295.

In summary, the conclusion is that it is not included in any of positions 295, 298, 302, 307, and 310 of the PCSK9 peptide or attached Substituent compounds are generally functional. A further conclusion is that amino acid substitutions at any of positions 295, 298, 302, and 310 are generally not attractive. As can be seen from Tables 3.1 and 3.2, nonetheless, the V307I mutation in combination with 301Leu appears to be acceptable or even attractive.

Taking further into consideration that peptides having amino acid substitutions at one of positions 295, 296, 298, 302, and 310 appear to have reduced functionality, while substitutions at 299, 315, and 320 appear to only slightly decrease functionality. This, on the other hand, also means that there may be a high degree of elasticity for the remaining amino acid residues, as Lys substitutions and attachment of side chains will affect these winners to the same extent as most other amino acid substitutions. Peptide.

    PCSK9 inhibitor with two substituents    

A series of compounds with two substituents are prepared. Double substitutions can be obtained by deuteration, alkylation, or combinations of N-terminus or Lys (K) residues. Again, the N-terminus can be an amino acid 293G or a variant amino acid residue, such as 292A, 293G, 293K, and 294T (in the example where the 293G line is deleted). These compounds are prepared with different substituents, although the two substituents on individual compounds are exactly the same. The main chain used in this study again included the N301L amino acid substitution combination N309R and various N-terminal and / or Lys substitutions (as needed) to obtain special tritiation / alkylation.

Once again, the inventors concluded that such substitutions were excellently tolerated based on various positions and combinations.

    More EGF (A) derivatives    

In order to further study the role of various amino acid substitutions in the EGF (A) sequence, more compounds were prepared and tested, as shown in Table 3.5.All compounds include one by substitution or extension by amino acid (using 333K) Substituted substituents for introduced Lys residues. All backbone peptides include one or more of N301L amino acid substitution and optionally N309R and I312E. All such substituents include fatty diacids containing 16-20 carbon atoms and linkers that are γGlu alone or extended with Ado-Ado and / or tranexamic acid (Trx) moieties.

The results in Table 3.5 above show that the internal wt lysine at position 312 can be substituted with Glu (E) and Gln (Q), Arg (R) or Asp (D). Based on this change, it is envisaged that a wide range of amino residues based on position 312 can be tolerated without interfering with the peptide's inhibitory function.

It also proved that several other amino acid substitutions are well tolerated, including G293N, T294G, D299A, N300H, H306Y, H306D, N309S, Q324G, and R329H, and as mentioned above, N295D and N300P are not attractive Acid substitution.

    PCSK9 binding containing GLP-1 analog and EGF (A) analog    

To further investigate whether PCSK9 binding functionality can be actively bound to GLP-1 receptor agonists, compounds containing GLP-1 analogs and EGF (A) analogs were tested in the same analysis and the results are included in the following table 3.6.

These data show that the compounds containing GLP-1 analog and EGF (A) analog maintain the PCSK9 binding activity associated with the EGF (A) analog of the compound. These data also show that there are only very few changes and that the orientation of the GLP-1 analog and the EGF (A) analog does not affect PCSK9 binding.

    C4-LDL uptake analysis in HepG2 cells    

An alternative assay for determining the inhibitory efficacy of PCSK9 peptides and their derivatives is to measure the uptake of LDL in HepG2 cells.

Analysis principle : LDL uptake is mainly mediated by endogenous hLDLR performance, and therefore LDL uptake capacity is an indirect measure of LDLR performance. hLDLR can be down-regulated in a dose-dependent manner by exogenous PCSK9 culture. Therefore, PCSK9 culture reduces the ability of cells to take up LDL molecules. This down-regulation of LDL uptake can then be antagonized by the addition of compounds that neutralize or inhibit PCSK9 / LDLR binding. Thus, the characteristics of PCSK9 inhibitors can be defined based on their ability to increase LDL uptake in the presence of PCSK9 and, for example, to offset PCSK9-mediated downregulation of hLDLR.

This analysis was performed using HepG2 cells (Sigma Aldrich ECACC: Acc no.85011430) grown in 10% lipoprotein-deficient fetal bovine serum (Sigma Aldrich # S5394) and measuring cell uptake of BODIPY fluorescently labeled LDL particles (Life Technologies Europe BV # L3483).

Analysis protocol : A 96-well plate (Perkin Elmer, ViewPlate-96 Black # 60005182) was dissolved at 37 ° C in a culture vessel with poly-D-lysine (10mg / L, Sigma Aldrich # P6407, dissolved in PBS (Gibco # 14190-094) Medium) for 1 hour. Next, the plates were washed 2 x in 100 μl PBS (Gibco # 14190-094). Test compositions for 8-point concentration curves of EGF (A) compounds were prepared, all of which were contained in analysis medium (DMEM (Gibco # 31966-021), 10% lipoprotein-deficient fetal bovine serum (Sigma Aldrich # S5394), and PCSK9 (10ug / ml) diluted in 1% Pen Strep (Cambrex # DE17-602E)) and added to the plate at a volume of 50ul / slot.

After 30-60 minutes, 50.000 HepG2 cells (Sigma-Aldrich: ECACC: Atcc no.85011430 lot: 13B023) diluted in the analysis medium will be added in a volume of 50 μl / well, and the plate will be in a CO2 permeable plastic (Antalis Team, LDPE bag 120 / 35x300x0,025mm # 281604) was cultured for 20 hours (at 37 ° C, 5% CO2). After that, empty the plate and immediately add 50 μl of FL-LDL (Life Technologies Europe BV # L3483) at a concentration of 10 μg / ml in the analysis medium to each slot, and incubate the plate in a CO2 permeable plastic bag for 2 hours (in 37 ° C, 5% CO2) and a black cover on the lid to protect it from light. The plate was emptied and washed twice with 100 μl of PBS (Gibco # 14190-094). Next, 100 μl of PBS (Gibco # 14190-094) was added and the plate was read on the SpecktraMax M4 (Molecular Probes, Invitrogen Detection Technologies) on the SpecktraMax M4 (Molecular Probes, Invitrogen Detection Technologies) using the following filters Ex (515nm) / Em (520nm) within 15 min. Read). EC50 values were calculated using GraphPad Prism, nonlinear regression curve fitting, sigmoidal dose-response (slope of change).

    The result    

LDL uptake analysis in HepG2 cells was performed as described above for a range of compounds.

The results are shown in Table 4.1 below. A lower EC50 value reflects a higher ability to reverse downregulation of PCSK9-mediated LDL uptake, and conversely an EC50 value indicates a compound with low ability to inhibit downregulation of PCSK9-mediated LDL uptake.

As can be seen, most compounds show an EC50 of 100-500 nM in the LDL uptake analysis, which indicates compounds with high ability to reverse PCSK9-mediated downregulation of LDL uptake (ie, increase LDL uptake).

LDL uptake was further evaluated for GLP-1 / EGF (A) compounds containing GLP-1 analogs and EGF (A) analogs and reconfirmed that the connection to GLP-1 analogs did not interfere with EGF (A) analog Functionality (see 4.2).

    C5-Pharmacokinetics (PK) in mini pigs    

The purpose of this study was to determine the in vivo time extension of GLP-1 derivatives after i.v. administration to mini-pigs, that is, it is equal to the time in the body (and thus their duration of action). This was done in a pharmacokinetic (PK) study in which the terminal half-life of the derivative of interest was determined. Terminal half-life means the time it takes to halve a certain plasma concentration during the terminal elimination period.

A female Göttingen mini pig line, about 8-12 months old and weighing about 20-30 kg, was obtained from the Ellegaard Göttingen Minipigs (Dalmose, Denmark) line for use in the study. Mini pigs were individually housed (pigs with permanent ducts) in a barn with straw as litter and the feeding system was restricted to Altromin 9030 mini pig feed (Altromin Spezialfutter GmbH & Co. KG) once daily.

Three weeks after acclimation, two permanent central venous catheters were implanted into the caudal vena cava of each animal. Animals were allowed to recover for 1 week after surgery, and then used for repeated pharmacokinetic studies with a proper clearance period between successive doses.

These derivatives were dissolved in a buffer containing 50 mM phosphate, 70 nM sodium chloride and 0.05% polysorbate 80, pH 7.4.

The intravenous injection of these derivatives (volume corresponding to 0.05 ml / kg and a dose of 2 nmol / kg) is given through a catheter, and blood is sampled at a predetermined time point until 14 days after administration (preferably from Additional catheters).

Blood samples (e.g., 0.8 ml) are collected into EDTA (8 mM) coated tubes and then centrifuged at 4 ° C and 1942 g for 10 minutes.

Plasma was pipetted into a Micronic tube on dry ice and kept at -20 ° C until the plasma concentration of the derivatives was analyzed using LOCI. Individual plasma concentration-time profiles were analyzed by non-compartment pharmacokinetics in Phoenix v.6.4 (Pharsight Inc., Mountain View, CA, USA) and the resulting terminal half-lives (harmonic mean) were determined.

    The result    

Pharmacokinetic research was performed using minipigs, as described above. Get the following results for the terminal half-life:

The compounds tested all have an increased terminal half-life compared to human GLP-1 (7-37).

Compound 21 containing a GLP-1 analogue with G at position 8 has a terminal half-life of 2 hours, which is 10-25 times shorter than that of other compounds with unnatural amino acids at position 8; Aib.

    C6-hPCSK9 challenge model    

The purpose of this study was to show the level of LDL receptor expression in mouse liver in response to the effect of inhibiting the effect of intravenously injected hPCSK9 with EGF (A) analogs or compounds comprising EGF (A) analogs as described herein The change.

    Method    

Healthy male BalBC or NMRI mice (Charles River, Germany) were injected sc or iv with EGF (A) analogs (or compounds containing EGF (A) analogs) and 0.4-0.4 minutes later in the tail vein. hPCSK9 (Sino Biologicals, China) was injected intravenously at a dose of mg / kg. Sixty minutes after the injection of hPCSK9, the animals were anesthetized in isoflurane and euthanized by cervical dislocation. The liver was then quickly cut out and rapidly frozen in liquid nitrogen. The liver was stored at -80 ° C until analysis.

    LDL-R Western Ink Dot Stain:    

Liver tissue samples (100 mg) were placed in 500 μl of a mixture of phosphatase inhibitors; PhosStop (Roche, 04 906 837 001) and a protease inhibitor mixture; compelate (Roche, 04 693 159 001) lysis buffer (Life Technology, FNN0011). After adding 1 steel ball, the tissue was homogenized at 30 Hz for 2.5 min. After centrifugation at 5000xg for 5 min, the total protein content was determined using a BCA protein analysis kit (Pierce, 23225). An equal amount of protein (60 μg) in sample buffer (Life Technology, NP0007) was boiled for 10 min and centrifuged at 14000 rpm for 2 min, and then loaded into a Criterion XT 3-8% Tris-acetate gel (BioRad # 345-0131) and accept SDS-PAGE. The protein was transferred to a nitrocellulose membrane (iBlot 2 NC Regular stacks, novex # IB23001) according to the manufacturer's instructions (Life Technology). Equal protein transfer was confirmed by staining the membrane with Ponceau S (Sigma, P7170) and the membrane was further blocked in blocking buffer (TBS-T, 2% Tween). The LDL-r protein was detected using a primary rabbit anti-LDLr antibody (Cayman Chemical Company # 10012422), and the β-actin protein was detected using a primary rabbit anti-β-actin antibody (abcam # ab6276). Both proteins were further developed with goat anti-rabbit secondary antibody (Biorad # 170-6516) complexed with peroxidase using Western Bright Quantum Chemiluminscent (Advansta # K-12042-D10) and photographed using a CCD camera (LAS3000, FujiFilm). Quantitative analysis of chemiluminescence signals from western blotting was performed using MultiGauge software (Fujifilm).

    The result    

The LDL-R performance level is measured by Western blotting and staining, and the performance level is compared. Performance was reduced by "vehicle-hPCSK9" (which represents the group injected with hPCSK9 alone). The group injected with the EGF (A) compound-hPCSK9 "showed that the performance of LDL-R was normalized because the performance returned to at least 90%.

These results show that hPCSK9 reduces the expression level of LDL-R and that this effect is suppressed by the EGF (A) compound tested. The data are presented in Tables 6.1 and 6.2 as a percentage change in the window between the baseline level (set to 100%) and the level down-regulated by hPCSK9 alone (set to 0%) in healthy control animals To outline. The six EGF (A) compounds tested were able to inhibit the effect of hPCSK9 on LDL-R expression levels and the levels of inhibition observed were similar to those observed using the control molecule Alirocumab.

    in conclusion    

Several compound examples have been shown to potentiate down-regulation of LDL-R expression levels through hPCSK9.

    Pharmacodynamics of C7-in db / db mice    

The purpose of this analysis was to demonstrate the acute efficacy of blood glucose (BG) and body weight (BW) in a diabetic setting.

The compounds were tested in a single dose study in a mouse model of obesity diabetes (db / db mice), as described below. The derivatives were tested at different doses (i.e., 0.3, 1.0, 3.0, 10, 30, and 100 nmol / kg or 1.0, 3.0, 10, 30, 100, and 300 nmol / kg). Denmark) (NIH31 (NIH 31M rodent feed, commercially available from Taconic Farms, Inc., US, see www.taconic.com) has been fed since birth) for research. After reaching the animal group, mice were given free access to standard food (eg Altromin 1324, Brogaarden, Gentofte, Denmark) and tap water and kept at 24 ° C. After 1-2 weeks of acclimation, baseline blood glucose was assessed twice a day. Only mice with a baseline blood glucose level of> 15 mM were included. Mice were assigned to treatment groups based on matched blood glucose levels and weight (N = 5-7 each group).

Animals were grouped to receive a vehicle (subcutaneous) or GLP-1 / PCSK9i derivative (0.3, 1.0, 3.0, 10, 30, or 100 nmol / kg or 1.0, 3.0, 10, 30, 100, and 300 nmol / kg) ( Subcutaneous), wherein the carrier is 50 mM sodium phosphate, 70 mM sodium chloride, 0.05% polysorbate 80, pH 7.4.

The GLP-1 / EGF (A) compound is dissolved in a carrier to administer a concentration of 0.05, 0.17, 0.5, 1.7, 5.0 or 17 nmol / mL or 0.17, 0.5, 1.7, 5.0, 17 or 50 nmol / ml. At the beginning of the experiment, animals were administered once at a dose-volume s.c. of 6 ml / kg (i.e. 300 μl per 50 g mouse).

On the day of dosing, blood glucose was assessed at time -½h in the morning, after which the mice were weighed. The GLP-1 / EGF (A) compound is administered at approximately time 0. On the day of dosing, blood glucose was evaluated at 1, 2, 4 and 8 hours after dosing.

On the following day, blood glucose was evaluated at time 24h, 48h, 72h, and 96h. On each day, mice were weighed after blood glucose sampling.

Mice were individually weighed on a digital scale.

Samples for measuring blood glucose were obtained from the tail microcapsules of awake mice. Blood (5 μl) was collected into heparin-treated capillaries and transferred to 250 μl glucose buffer (EKF System solution, Eppendorf, Germany). Glucose oxidase method (glucose analyzer Biosen 5040, EKF Diagnostic, GmbH, Palebben, Germany) was used to measure glucose concentration. The samples were maintained at room temperature for up to 1 h or at 4 ° C for a maximum of 24 h until analysis.

Blood glucose minus baseline and weight minus baseline were calculated in mice.

    The result    

GLP-1 / EGF (A) compounds 1, 2, 21, 22, 23, 25, 26, 27, 29, and 32 were tested in single-dose studies, as described above. These derivatives are available in different doses (i.e. 0.3, 1.0, 3.0, 10, 30 and 100 nmol / kg (compounds 2, 21, 22, 23, 25 and 26) or 1.0, 3.0, 10, 30, 100 and 300 nmol / kg (compounds 1, 27, 29 and 32)).

Table 7.1 gives a summary of the maximum efficacy (Emax) for the highest doses of delta blood glucose and delta weight at 24 hours after dosing. If the two highest dose levels do not give similar efficacy and therefore the true Emax may not have been reached, the values are marked with an asterisk (*).

In order to obtain the indication of the efficacy of GLP-1 / PCSK9i derivatives on blood glucose and body weight, the area under the curve of △ blood glucose (AUC △ BG _24h ) from 0 to 24 hours and the △ weight at 24 hours after administration were calculated (△ BW _24h ). Based on the dose-response curves of these parameters, an effective dose of 50% was calculated for AUC ΔBG _24h and ΔBW _24h (ED50, the dose of GLP-1 derivative giving the intermediate response at baseline and maximum efficacy). ED50 can be used as an estimate of the potency of GLP-1 / PCSK9i derivatives. The following results were obtained (average of all individual determinations).

All the compounds tested showed a dose-dependent reduction of blood glucose and in vivo efficacy in vivo.

Although certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those skilled in the art. Therefore, it should be understood that the scope of the appended patent application is intended to cover all such modifications and changes that fall within the true spirit of the invention.

    C8-Pharmacodynamic Study in DIO Rats    

The purpose of this analysis was to demonstrate the subchronic efficacy of body weight (BW) and total cholesterol levels in an obesity setting. The compounds were tested in a feed-induced obesity (DIO) rat model in a subchronic dose study for 21 days, as described below. Derivatives were tested at different doses (i.e., 30 and 300 nmol / kg, and in some instances the 300 nmol / kg group was dosed with a higher dose of 900 nmol / kg for the time indicated).

Shi-Dao rats (from Charles River, France) (fed with 60% high fat diet (D12492, commercially available from Research Diets, Inc) from 6 weeks old) arrived at my animal at 22 weeks old group. After reaching the animal group, rats were allowed free access to 45% high-fat diet (D12451, commercially available from Research Diets, Inc) and tap water and rats were placed under controlled light (12h: 12h light / dark cycle; light Open at 06: 00-18: 00) and temperature (22 ± 2 ℃). After 2-3 weeks of acclimation, rats were assigned to the treatment group based on the matched body weight and fat percentage (N = 10 per group).

Animals were grouped to receive a vehicle (subcutaneous) or GLP-1 / EGF (A) compound (30 or 300 nmol / kg, in some examples rats from the 300 nmol / kg group received 900 nmol / kg of the indicated number of days ) (Subcutaneous), wherein the carrier is 50 mM phosphate, 70 mM sodium chloride, 0.007% polysorbate 20, pH 7.4. The GLP-1 / EGF (A) compound is dissolved in a carrier to administer 15 (for upward adjustment), 50 (for upward adjustment), 150, 500 (for upward adjustment), or 1500 nmol / ml concentration.

Animals were administered subcutaneously once a day for 22 days in the morning at a dosage of 0.2 ml / kg. The dose was adjusted slowly upwards so that rats received 3 nmol / kg on the first day, 10 nmol / kg on the second day, 30 nmol / kg on the third day, and 100 nmol / kg if appropriate on the fourth day and on the first day. 300nmol / kg for five days. The 30nmol / kg group received the full dose from the third day until the end of the experiment. The 300nmol / kg group received the full dose from the fifth day until the end of the experiment. Rats administered with GLP-1 / EGF (A) compound 41 at 300 nmol / kg received 900 nmol / kg from the 16th day until the end of the experiment. Rats administered at 300 nmol / kg of GLP-1 / EGF (A) compound 48 received 900 nmol / kg from the 20th day until the end of the experiment. The 900 nmol / kg dose was achieved by increasing the dosing volume of the 1500 nmol / ml solution to 0.6 ml / kg.

Rats were weighed daily on a digital scale immediately before administration. Food container weights are also weighed daily to calculate food intake. Body composition was assessed by MR scan (Echo MRI 700, Houston, TX USA) 3 to 4 days before the start of dosing and on the 20th or 21st. Sublingual blood samples were obtained from conscious rats 5 days before the start of dosing and at the end of the study. Blood samples were collected in EDTA tubes and mixed thoroughly by inversion. Immediately after collection, the EDTA tube was placed on ice. The EDTA blood sample was centrifuged at 6000G for 5 min at 4 ° C, and the plasma sample was stored at -80 ° C until analysis. The samples were analyzed for total cholesterol levels on a Cobas analyzer (Cobas 6000, Roche Diagnistics, USA).

Baseline minus body weight and total cholesterol level minus baseline were calculated for each rat and averaged for each group.

    The result    

GLP-1 / EGF (A) compounds 41, 48, and 69 were tested in a subchronic dose study, as described above. Derivatives are given at different doses (i.e. 30 and 300 nmol / kg (GLP-1 / EGF (A) compound 69) or 30 and 300 nmol / kg and the last 2 days (GLP-1 / EGF (A) compound 41) or last On the 7th (GLP-1 / EGF (A) compound 48) the dose was increased to 900 nmol / kg).

Table 8.1 gives an overview of the average body weight (mean ± SEM) compared to baseline body weight and the average delta (mean ± SEM) of plasma total cholesterol levels compared to baseline in each group as a percentage.

    C9- chemical stability    

Formulations of GLP-1 / EGF (A) compounds 69 and 313 were prepared to study the potential stabilizing efficacy (reduction of isomer formation) of EGF (A) analogs in which 321D was substituted with 321E. The compound concentration in a formulation consisting of 20 mM Tris, pH 7.4, 18.4 mg / ml propylene glycol, and 0.43 mM CaCl ₂ was 2 mg / mL. These formulations are prepared by dissolving the freeze-dried material at a final concentration into MQ water containing Tris, propylene glycol, and CaCl ₂ . The pH was adjusted using 0.1N HCl (aq) and 0.1N NaOH (aq). Each formulation was sterile filtered and filled on HPLC glass vials and stored statically in a temperature controlled cabinet at 37 ° C. Samples were drawn from the HPLC vials at selected time points (times 0, 1 week, 2 weeks, 4 weeks) and frozen for subsequent UPLC-MS analysis.

Stability indication using a BEH C4 column (300Å, 1.7um, 1.0x150mm, Waters) and 0.1% formic acid (solution A) in water / 0.1% formic acid (solution B) in acetonitrile The purity method evaluates the loss of purity of a formulation under thermal stress. The following conditions were used: column temperature: 50 ° C; flow rate: 0.30mL / min; wavelength of UV detector: 215nm. The gradient is from 31% to 39% B over a period of 41 minutes. The LC streamline was linearly injected into an Orbitrap Fusion Lumos mass spectrometer (Thermo Fischer Scientific) equipped with an electrospray interface operating in cation mode. This purity method was shown to be compatible with the previously mentioned formulations and no content / analog loss was observed. The amount of isomers formed was determined by mass-based extraction from total ion chromatograms of various samples (i.e., samples that were cultured at time 0 and at 37 ° C for 2 and 4 weeks), The percentage of isomers is calculated from the integral of the isomer peak area to the main peak (API) area.

The results in Table 9.1 show that replacing 321D with 321E significantly reduced the amount of isomer formation from 20.2% to 4.2% after incubation at 37 ° C for 4 weeks.

Although certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those skilled in the art. Therefore, it should be understood that the scope of the appended patent application is intended to cover all such modifications and changes that fall within the true spirit of the invention.

<110> Danish Commercial Norfolk. Londick co., Ltd.

<120> Bifunctional compounds

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<220>

<223> Synthesis

<400> 99

<210> 100

<211> 40

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<400> 100

<210> 101

<211> 40

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<400> 101

<210> 102

<211> 40

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<400> 102

<210> 103

<211> 41

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<400> 103

<210> 104

<211> 41

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<400> 104

<210> 105

<211> 41

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<400> 105

<210> 106

<211> 40

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<400> 106

<210> 107

<211> 40

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<400> 107

<210> 108

<211> 40

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<400> 108

<210> 109

<211> 40

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<400> 109

<210> 110

<211> 40

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<400> 110

<210> 111

<211> 40

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<400> 111

<210> 112

<211> 40

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<400> 112

<210> 113

<211> 40

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<400> 113

<210> 114

<211> 40

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<400> 114

<210> 115

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<400> 115

<210> 116

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<400> 116

<210> 117

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<400> 117

<210> 118

<211> 24

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<400> 118

<210> 119

<211> 40

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<400> 119

<210> 120

<211> 80

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<400> 120

<210> 121

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<400> 121

<210> 122

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<400> 122

<210> 123

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<400> 123

<210> 124

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<400> 124

<210> 125

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<400> 125

<210> 126

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<400> 126

<210> 127

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<400> 127

<210> 128

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<400> 128

<210> 129

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<400> 129

<210> 130

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<400> 130

<210> 131

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<400> 131

<210> 132

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<400> 132

<210> 133

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<400> 133

<210> 134

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<400> 134

<210> 135

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<400> 135

<210> 136

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<400> 136

<210> 137

<211> 31

<212> PRT

<213> wise man

<400> 137

<210> 138

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 138

<210> 139

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 139

<210> 140

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<400> 140

<210> 141

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<400> 141

<210> 142

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 142

<210> 143

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 143

<210> 144

<211> 26

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 144

<210> 145

<211> 27

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 145

<210> 146

<211> 28

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 146

<210> 147

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 147

<210> 148

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 148

<210> 149

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 149

<210> 150

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 150

<210> 151

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 151

<210> 152

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 152

<210> 153

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 153

<210> 154

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 154

<210> 155

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 155

<210> 156

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 156

<210> 157

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 157

<210> 158

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 158

<210> 159

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 159

<210> 160

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 160

<210> 161

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 161

<210> 162

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 162

<210> 163

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 163

<210> 164

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 164

<210> 165

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 165

<210> 166

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 166

<210> 167

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 167

<210> 168

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 168

<210> 169

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 169

<210> 170

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 170

<210> 171

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 171

<210> 172

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 172

<210> 173

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 173

<210> 174

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 174

<210> 175

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 175

<210> 176

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 176

<210> 177

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 177

<210> 178

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 178

<210> 179

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 179

<210> 180

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 180

<210> 181

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 181

<210> 182

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 182

<210> 183

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 183

<210> 184

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 184

<210> 185

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 185

<210> 186

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 186

<210> 187

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> Variant

<222> (2) .. (2)

<223> where X is A, G, W, or Aib

<220>

<221> Variant

<222> (6) .. (6)

<223> where X is F or K

<220>

<221> Variant

<222> (15) .. (15)

<223> where X is E, G, or K

<220>

<221> Variant

<222> (17) .. (17)

<223> where X is Q, G, or K

<220>

<221> Variant

<222> (18) .. (18)

<223> where X is A, G, V or K

<220>

<221> Variant

<222> (19) .. (19)

<223> where X is A, G, V or K

<220>

<221> Variant

<222> (20) .. (20)

<223> where X is K or R

<220>

<221> Variant

<222> (21) .. (21)

<223> where X is E, G, or K

<220>

<221> Variant

<222> (23) .. (23)

<223> where X is I, A, or V

<220>

<221> Variant

<222> (24) .. (24)

<223> where X is A, G, or K

<220>

<221> Variant

<222> (25) .. (25)

<223> where X is W, G, or K

<220>

<221> Variant

<222> (26) .. (26)

<223> where X is L, G, T, V, I, or K

<220>

<221> Variant

<222> (27) .. (27)

<223> Where X is V, G, I, L, K or does not exist

<220>

<221> Variant

<222> (28) .. (28)

<223> where X is K, R, Q or does not exist

<220>

<221> Variant

<222> (29) .. (29)

<223> Where X is G or does not exist

<220>

<221> Variant

<222> (30) .. (30)

<223> where X is R, K or does not exist

<220>

<221> Variant

<222> (31) .. (31)

<223> Where X or G does not exist

<400> 187

<210> 188

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 188

<210> 189

<211> 75

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 189

<210> 190

<211> 80

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 190

<210> 191

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 191

<210> 192

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 192

<210> 193

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 193

<210> 194

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 194

<210> 195

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 195

<210> 196

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 196

<210> 197

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 197

<210> 198

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 198

<210> 199

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 199

<210> 200

<211> 83

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 200

<210> 201

<211> 95

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 201

<210> 202

<211> 111

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 202

<210> 203

<211> 151

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 203

<210> 204

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 204

<210> 205

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 205

<210> 206

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 206

<210> 207

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 207

<210> 208

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 208

<210> 209

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 209

<210> 210

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 210

<210> 211

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 211

<210> 212

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 212

<210> 213

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 213

<210> 214

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 214

<210> 215

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 215

<210> 216

<211> 81

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 216

<210> 217

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 217

<210> 218

<211> 81

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 218

<210> 219

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<400> 219

<210> 220

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<400> 220

<210> 221

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 221

<210> 222

<211> 73

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 222

<210> 223

<211> 74

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 223

<210> 224

<211> 75

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 224

<210> 225

<211> 76

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 225

<210> 226

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 226

<210> 227

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 227

<210> 228

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 228

<210> 229

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 229

<210> 230

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 230

<210> 231

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 231

<210> 232

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 232

<210> 233

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 233

<210> 234

<211> 80

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 234

<210> 235

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 235

<210> 236

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 236

<210> 237

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 237

<210> 238

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 238

<210> 239

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 239

<210> 240

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 240

<210> 241

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 241

<210> 242

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 242

<210> 243

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 243

<210> 244

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 244

<210> 245

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 245

<210> 246

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 246

<210> 247

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 247

<210> 248

<211> 80

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 248

<210> 249

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 249

<210> 250

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 250

<210> 251

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 251

<210> 252

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 252

<210> 253

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 253

<210> 254

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 254

<210> 255

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 255

<210> 256

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 256

<210> 257

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 257

<210> 258

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 258

<210> 259

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 259

<210> 260

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 260

<210> 261

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 261

<210> 262

<211> 80

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 262

<210> 263

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 263

<210> 264

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 264

<210> 265

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 265

<210> 266

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 266

<210> 267

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 267

<210> 268

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 268

<210> 269

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 269

<210> 270

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 270

<210> 271

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 271

<210> 272

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 272

<210> 273

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 273

<210> 274

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 274

<210> 275

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 275

<210> 276

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 276

<210> 277

<211> 80

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 277

<210> 278

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 278

<210> 279

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 279

<210> 280

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 280

<210> 281

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 281

<210> 282

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 282

<210> 283

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 283

<210> 284

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 284

<210> 285

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 285

<210> 286

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 286

<210> 287

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 287

<210> 288

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 288

<210> 289

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 289

<210> 290

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 290

<210> 291

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 291

<210> 292

<211> 80

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 292

<210> 293

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 293

<210> 294

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 294

<210> 295

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 295

<210> 296

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 296

<210> 297

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 297

<210> 298

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 298

<210> 299

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 299

<210> 300

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 300

<210> 301

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 301

<210> 302

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 302

<210> 303

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 303

<210> 304

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 304

<210> 305

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 305

<210> 306

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 306

<210> 307

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 307

<210> 308

<211> 80

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 308

<210> 309

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 309

<210> 310

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 310

<210> 311

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 311

<210> 312

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 312

<210> 313

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 313

<210> 314

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 314

<210> 315

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 315

<210> 316

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 316

<210> 317

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 317

<210> 318

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 318

<210> 319

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 319

<210> 320

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 320

<210> 321

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 321

<210> 322

<211> 80

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 322

<210> 323

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 323

<210> 324

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 324

<210> 325

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 325

<210> 326

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 326

<210> 327

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 327

<210> 328

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 328

<210> 329

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 329

<210> 330

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 330

<210> 331

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 331

<210> 332

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 332

<210> 333

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 333

<210> 334

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 334

<210> 335

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 335

<210> 336

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 336

<210> 337

<211> 80

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 337

<210> 338

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 338

<210> 339

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 339

<210> 340

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 340

<210> 341

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 341

<210> 342

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 342

<210> 343

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 343

<210> 344

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 344

<210> 345

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 345

<210> 346

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 346

<210> 347

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 347

<210> 348

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 348

<210> 349

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 349

<210> 350

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 350

<210> 351

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 351

<210> 352

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 352

<210> 353

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 353

<210> 354

<211> 80

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 354

<210> 355

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 355

<210> 356

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 356

<210> 357

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 357

<210> 358

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 358

<210> 359

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 359

<210> 360

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 360

<210> 361

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 361

<210> 362

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 362

<210> 363

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 363

<210> 364

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 364

<210> 365

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 365

<210> 366

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 366

<210> 367

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 367

<210> 368

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 368

<210> 369

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 369

<210> 370

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 370

<210> 371

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 371

<210> 372

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 372

<210> 373

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 373

<210> 374

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 374

<210> 375

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 375

<210> 376

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 376

<210> 377

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 377

<210> 378

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 378

<210> 379

<211> 80

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 379

<210> 380

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 380

<210> 381

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 381

<210> 382

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 382

<210> 383

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 383

<210> 384

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 384

<210> 385

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> GTNECLDNLGGCSHVCRDLEIGYECLCPEGFQLVAQRRCEGQAPGQAPHXEGTFTSDVSSYLEGQ AAKEFIAWLVKGRG

<222> (50) .. (50)

<223> Aib

<220>

<221> MOD_RES

<222> (50) .. (50)

<223> Aib

<400> 385

<210> 386

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (50) .. (50)

<223> Aib

<400> 386

<210> 387

<211> 79

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<400> 387

<210> 388

<211> 111

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Aib

<400> 388

Claims (16)

    A compound comprising a GLP-1 analog and an EGF (A) analog, wherein i. The GLP-1 analog is an analog of GLP-1 (7-37) identified by SEQ ID NO: 137 and ii The EGF (A) analog is an analog of the EGF (A) domain (293-332) of LDL-R identified by SEQ ID No: 1.         A compound according to claim 1, wherein the compound is bifunctional.         The compound of claim 1, wherein the compound comprises a fusion polypeptide comprising the GLP-1 analog and the EGF (A) analog.         The compound according to claim 3, wherein the fusion polypeptide comprises the GLP-1 analog at the N-terminus and the EGF (A) analog at the C-terminus.         The compound of claim 3 or claim 4, wherein the fusion polypeptide comprises a peptide spacer, such as a spacer selected from the group of spacers defined by SEQ ID NOs. 115-136.         A compound according to any one of the preceding claims, wherein the compound comprises one or two Lys residues.         The compound of any one of the preceding claims, wherein the GLP-1 analog is selected from the group of GLP-1 analogs defined by: SEQ ID NOs 138 to 187, such as SEQ ID NO .: 139-146, 155-162, and 164-173, such as SEQ ID NO .: 139-142, 155-162, and 164-173, such as SEQ ID NO .: 139, 142, 155-162, and 164-173, such as SEQ ID NO .: 139, 155-162 and 164-173, such as SEQ ID NO .: 155-162 and 164-173, such as SEQ ID NO .: 139 and 164 or such as SEQ ID NO .: 139 or 164.         The compound of any one of the preceding claims, wherein the EGF (A) analog is selected from the group of EGF (A) analogs defined by: SEQ ID NOs 2 to 114, such as SEQ ID NO .2-4, 6-19, 21-44, 46, 47, 49-53, 55, and 58-114, such as SEQ ID NO: 19, 21, 73, 107, 108, 109, 110, 111, 112, 113 and 114, such as SEQ ID NOs: 107 and 108 or such as SEQ ID NO .: 108.         The compound of any one of the preceding claims, wherein the fusion polypeptide is selected from the group consisting of a sequence defined by SEQ ID NOs 188-384 and 387-388.         A compound according to any one of the preceding claims, wherein the compound comprises at least one half-life extending substituent.         A compound according to any one of the preceding claims, wherein the compound comprises at least one substituent comprising a fatty acid group and a linker.         The compound of claim 11 or claim 12, wherein the at least one substituent is attached through a lys residue.         A compound selected from the group of compounds defined as GLP-1 / EGF (A) compounds # 41, # 48, # 69, and # 306, such as # 306 and # 69, or compounds such as # 306 or # 69.         A compound according to any one of the preceding claims for use in a method of treatment.         A compound according to any one of the preceding claims for use in the treatment of diabetes, overweight and / or cardiovascular diseases.         A method of treating diabetes, overweight and / or cardiovascular disease, comprising administering a pharmaceutically effective dose of a compound according to any one of claims 1-14 to a patient in need thereof.